Methods and devices for in situ formed nerve cap

ABSTRACT

Disclosed are methods, devices and materials for the in situ formation of a nerve cap to inhibit neuroma formation following planned or traumatic nerve injury. The method includes the steps of identifying a severed end of a nerve, and positioning the severed end into a cavity defined by a form. A transformable media is introduced into the form cavity to surround the severed end. The media is permitted to undergo a transformation from a first, relatively flowable state to a second, relatively non flowable state to form a protective barrier surrounding the severed end. The media may be a hydrogel, and the transformation may produce a synthetic crosslinked hydrogel protective barrier. The media may include at least one anti-regeneration agent to inhibit nerve regrowth

INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) as a bypasscontinuation of PCT App. No. PCT/US2019/040429 filed on Jul. 2, 2019,which in turn is a nonprovisional application of U.S. Prov. App. No.62/692,858 filed on Jul. 2, 2018 and U.S. Prov. App. No. 62/822,881filed on Mar. 24, 2019, each of the foregoing of which are herebyincorporated by reference in their entireties.

BACKGROUND

Neuromas are benign tumors that arise from neural tissue and arecomposed of abnormally sprouting axons, Schwann cells, and connectivetissue. Even though neuromas can appear following various types ofinjuries, some of the most common and challenging to treat are derivedfrom trauma or surgical procedures in which neural tissue was damaged ortransected. Amputation surgeries necessitate the transection of one ormore sensory or mixed nerves. Chronic neuropathic pain, attributed toneuroma formation, develops in up to 30% of patient's post-surgery andresults in downstream challenges with wearing a prosthesis. In additionto traumatic and amputation related neuromas, neuromas form acrossmultiple clinical indications such as in general surgery (hernia repair,mastectomy, laparoscopic cholecystectomy), gynecologic surgery(C-section, hysterectomy), and orthopedics (arthroscopy, amputation,knee replacement).

Neuromas develop as a part of a normal reparative process followingperipheral nerve injury. They are formed when nerve recovery towards thedistal nerve end or target organ fails and nerve fibers improperly andirregularly regenerate into the surrounding scar tissue. Neuromasinclude a deranged architecture of tangled axons, Schwann cells,endoneurial cells, and perineurial cells in a dense collagenous matrixwith surrounding fibroblasts (Mackinnon S E et al. 1985. Alteration ofneuroma formation by manipulation of its microenvironment. PlastReconstr Surg. 76:345-53). The up regulation of certain channels andreceptors during neuroma development can also cause abnormal sensitivityand spontaneous activity of injured axons (Curtin C and Carroll I. 2009.Cutaneous neuroma physiology and its relationship to chronic pain. J.Hand Surg Am. 34:1334-6). Haphazardly arranged nerve fibers are known toproduce abnormal activity that stimulates central neurons (Wall P D andGutnick M. 1974. Ongoing activity in peripheral nerves; physiology andpharmacology of impulses originating from neuroma. Exp Neurol.43:580-593). This ongoing abnormal activity can be enhanced bymechanical stimulation, for example, from the constantly rebuilding scarat the injury site (Nordin M et al. 1984. Ectopic sensory discharges andparesthesia in patients with disorders of peripheral nerves, dorsalroots and dorsal columns. Pain. 20:231-245; Scadding J W. 1981.Development of ongoing activity, mechanosensitivity, and adrenalinesensitivity in severed peripheral nerve axons. Exp Neurol. 73:345-364).

Neuromas of the nerve stump or neuromas-in-continuity are unavoidableconsequences of nerve injury when the nerve is not, or cannot be,repaired and can result in debilitating pain. It has been estimated thatapproximately 30% of neuromas become painful and problematic. This isparticularly likely if the neuroma is present at or near the skinsurface as physical stimulation induces signaling in the nerve resultingin a sensation of pain.

The number of amputees in the world has risen significantly in recentyears, with war injuries and dysvascular diseases such as diabetesaccounting for approximately 90% of all amputee cases. There arecurrently about 1.7 million amputees living in the United States alone,and over 230,000 new amputee patients are discharged annually fromhospitals. Further, it has been estimated that there will be a 20%increase in the number of new amputee cases per year by 2050.

Unfortunately, due to persistent pain in limb remnants, about 25% ofamputees are not able to commence rehabilitation, much less resumeordinary daily activities. The cause of such pain can be a neuroma. Onerecent study reported that 78% of amputees experienced mild to severepain as a consequence of neuroma formation over the 25-year studyperiod, of which 63% described the pain as constant aching pain. Thepain is also frequently described as sharp, shooting, or electrical-likephantom sensations that persist for years after surgical amputation. Inaddition, patients experience tenderness to palpation of the skinoverlying the neuroma, spontaneous burning pain, allodynia, andhyperalgesia.

While various methods have been used to prevent, minimize, or shieldneuromas in an attempt to minimize neuropathic pain, the currentclinical “gold standard” for treating neuromas is traction neurectomy,in which the nerve is pulled forward under traction and transected asfar back as possible in the hope that, if a neuroma forms, that it willbe located deep in the tissue. Another well recognized approach is tobury the proximal nerve end (that will form the neuroma) into muscle ora hole drilled in bone. The nerve is then sutured to the muscle orperiosteum of the bone to maintain its position. The rationale for thisis that the surrounding tissue cushions and isolates the neuroma toinhibit stimulation and the resulting painful sensations. However, thisprocedure can greatly complicate surgery, as significant additionaldissection of otherwise healthy tissue is required to place the nervestump. This, coupled with poor and variable efficacy, the lack ofappropriate/available tissue, and the additional procedural timerequired, result in the procedure being rarely performed to preventneuroma formation.

Another method is to cut the nerve stump back to leave a segment orsleeve of overhanging epineurium. This overhang can be ligated to coverthe face of the nerve stump. Alternatively, a segment of epineurium canbe acquired from other nerve tissue or a corresponding nerve stump canbe cut back to create an epineurium sleeve that can be used to connectwith and cover the other nerve stump.

Yet another method that is commonly used is a suture ligation, where aloop of suture is placed around the end of the nerve and tightened. Thispressure is believed to mechanically block the exit of axons and causesthe terminal end to eventually form scar tissue over the site. Clinicaland pre-clinical evidence has shown, however, that this procedure cancause a painful neuroma to form behind the ligation. Furthermore, theligated nerve is generally not positioned to minimize mechanicalstimulation of the neuroma, since it is anticipated that the scar tissuewill provide sufficient protection to the nerve end.

Other methods used clinically include placing the nerve stump within asolid implantable silicone or biodegradable polymer tube with an open,or more recently, a sealed end (e.g. Polyganics NEUROCAP); wrapping theproximal nerve end with a harvested vein or fat graft, again with thegoal of providing a physical barrier to aberrant nerve regeneration. Theuse of biomaterial implant devices and methods necessitate insertion andsecuring the nerve with sutures in the opening of the device, which canbe difficult and further damage the nerve end. For example, the currentprocedure for securing the NEUROCAP requires a suture be placed in theepineurium of the nerve and through the wall of the tube and followed bypulling and stuffing the nerve into the lumen of the tube using thesuture and the placement of several sutures to retain the nerve in thedevice. These methods and devices can also result in mechanicalstimulation of the neuroma tissue as a result of 1) mismatch between thetissue compliance and the rigidity of the conduit and 2) inability ofthe cap to prevent neuroma formation within the cap, with resultingsensation of pain. Although these nerve caps degrade over a period of 3months to 18 months, substantial degradation-mediated mass loss occursin the first three to six months resulting in the exposure of atemporarily protected neuroma to the surrounding environment. Thus, theefficacy of these solid implantable caps is limited by the ability ofthe cap to conform to the proximal end of the nerve and prevent neuromaformation and secondarily their subsequent degradation to expose theneuroma to the surrounding environment. Finally, since these methodsrequire suturing using fine sutures (9-0 nylon) the procedural time andskill required to secure these implants under surgical magnification(loupes) or harvested tissue prohibits surgeons from more broadlyadopting these procedures.

Unfortunately, current methods for addressing the formation of and paincaused by neuromas have not been widely adopted. The need thereforeremains for an effective technology or therapy for controlling orinhibiting neuroma formation following inadvertent or planned surgicalor traumatic nerve injury in addition to reducing scar formation andperineural adhesions.

A variety of biomaterial conduits have been explored preclinically totry to prevent neuroma formation, including other solid implantablebiodegradable polymeric conduits based on polylactide/polycaprolactone(Onode et al (2019) Nerve capping with a nerve conduit for the treatmentof painful neuroma in the rat sciatic nerve, J Neurosurg. p. 1-9; Yan etal (2014) Mechanisms of Nerve Capping Technique in Prevention of PainfulNeuroma Formation, PLOS One, 9(4) p. 1-11; Yi et al (2018) PainfulTerminal Neuroma Prevention by Capping PGRD/PDLLA Conduit in Rat SciaticNerves Adv. Sci, 1-11), atelocollagen (Sakai et al (2005) Prevention andTreatment of Amputation Neuroma by an Atelocollagen Tube in Rat SciaticNerves. J Biomed Mater Res Part B: Appl Biomater 73B: 355-360) orporcine small intestine submucosa (Tork et al (2018), ePoster:Prevention of Neuromas with a Porcine SIS Nerve Cap: HistopathologicEvaluation,http://meeting.handsurgery.org/files/2018/ePosters/HSEP106.pdf) ormicrocrystalline chitosan (Marcol et al (2011) Reduction ofPost-Traumatic Neuroma and Epineural Scar Formation in Rat Sciatic Nerveby Application of Microcrystallic Chitosan. Microsurgery, 31: 642-649).These approaches have not been successful to date in preventing theformation of neuromas either because, again, the solid implants do notform in situ and create a potential space to permit nerve outgrowth andvarying degrees of neuroma formation or, critically, because the in vivopersistence of the materials was not sufficient to prevent neuromaformation.

Some embodiments as disclosed here have been demonstrated to preventneuroma formation preclinically and 1) eliminate the need for suturing,dragging or stuffing of the nerve inside a conduit, 2) conform to theend of the nerve stump providing a physical barrier to nerveregeneration, and 3) provide mechanical strength to prevent nerveregeneration for a period of two months, preferably three months or morenecessary to prevent nerve outgrowth during the growth regenerativephase after nerve injury. In situ forming implants described herein canbe compliant with the surrounding tissue, adhere to but do not compressthe underlying nerve tissue, are flexible such that they can move overregions of tissue involving joints or where nerves slide relative toother tissues and prevent scar tissue and adhesions forming aroundnerves. Finally, some of these in situ forming implants can be deliveredwithout advanced surgical training. In other situations, there remains aneed for a technology that prevent nerve outgrowth into the surroundingtissue and direct the outgrowth of a transected or compressed nerve intothe distal nerve stump or allograft/autograft. With this, in someaspects, a suture-free technology that can direct nerve regenerationfrom a proximal nerve stump directly (via direct coaptation/anastomoseswith distal nerve stump) or indirectly (through a nerve conduit,guidance channel, allograft, autograft), or through a growth-permissivematrix into the distal nerve stump is described. In addition, in someaspects, a technology that allows detensioning of the anastomoses siteis described to promote better nerve regeneration. Lastly, in someaspects, a technology that can be quickly and broadly applied to nervesto prevent inadvertent damage to adjacent nerves during a variety ofsurgical procedures is desirable.

SUMMARY

There is provided in accordance with one aspect of the present inventiona method of in situ formation of a conforming, protective nerve cap toinhibit neuroma formation at a severed nerve end. The method comprisesthe steps of identifying a severed end of a nerve; positioning thesevered end into a cavity defined by a form; introducing media into theform to surround the severed end; and permitting the media to undergo atransformation from a first, relatively flowable state to a second,relatively non flowable state to form a protective conforming barriersurrounding the severed end. The method may further comprise the step ofremoving the form, to leave behind a formed biocompatible in situprotective nerve cap.

The identifying a severed end of a nerve step may comprise identifying anerve severed such as by cutting or ablation, or severed traumatically.The form may comprise a nerve guide, and the positioning step maycomprise positioning the nerve such that the nerve guide maintains thesevered end within the cavity spaced apart from a sidewall of the form.The severed end may be positioned at least about 0.1 mm or 2 mm awayfrom the sidewall or more preferably about 1 mm away. Preferably theform is either bioresorbable or is composed of a flexible nondegradablematerial that can easily be removed from the surgical site afterformation of the in situ nerve cap.

The transformation from flowable to nonflowable state may occur withinabout 1 minute, or within about 30 seconds or within about 10 seconds ofthe introducing step. The method may additionally comprise the step ofblotting a volume of axoplasm from the severed nerve prior to theintroducing step.

In one implementation of the invention, the form may comprise a firstconfiguration in which the cavity is exposed, and a second configurationin which the cavity is partially or completely covered; and furthercomprising the step of advancing the form from the first configurationto the second configuration following the introducing nerve step.Alternatively, the step of advancing the form from the firstconfiguration to the second configuration may occur prior to theintroducing the nerve or media steps. The form may alternativelycomprise an open cell foam, and the cavity comprises a tortuous,interconnected interstitial volume within the foam. In the latterembodiment, the form would remain in place in situ after integrationwith and formation of the nerve cap.

The identifying a severed nerve step may include the step of severing atarget nerve. The step may additionally comprise transecting the nervecleanly at an oblique angle prior to placing the nerve within the form.The transformation step may comprise a crosslinking reaction or apolymerization and the use of an in situ forming hydrogel that canintercalate with the host tissue to form an adhesion between thehydrogel and the tissue. In the preferred embodiment, the hydrogel is aneutral or negatively charged material with submicron or smaller poreswhich permit nutrient and protein exchange but not cellularinfiltration. In one implementation, the transformation produces asynthetic crosslinked hydrogel protective barrier through which nervescannot regenerate around the end of a transected nerve stump.

The use of PEG as a biomaterial for delivery to nerves is well known inthe art. Increasingly, there is an appreciation that biomaterials,including PEG hydrogels, need to be tuned for specific applications.Properties, including molecular weight, degradation kinetics, PEG shape(linear vs multi-arm vs dendritic), degree of crosslinking, degree ofsubstitution, crosslinking type (electrophilic-nucleophilic or freeradical), gelation time, arm length (in the case of multi-arm PEGs),functional groups, hydrolytic linkages, and other factors such as pH andbuffer selection are tailored for specific applications directed towardsnerves to prevent neuroma formation.

In some embodiments, disclosed herein is a dual component in situforming biomaterial composition comprising a nerve growth permissivecomponent and a nerve growth inhibitory component.

In some embodiments, the nerve growth permissive component is deliveredfirst and the nerve growth inhibitory component delivered second.

In some embodiments, the nerve growth permissive components conform tothe nerve and facilitate nerve ingrowth into, through and across thebiomaterial into the distal stump.

In some embodiments, the nerve growth inhibitory components preventsnerve growth into the material.

In some embodiments, the nerve growth inhibitory components acts as aguide upon which nerve regeneration can occur.

In some embodiments, the biomaterial components comprise an in situforming gel.

In some embodiments, the biomaterial components comprise in situ formingcrosslinked gel, microparticles, nanoparticle, slurry or micelles.

In some embodiments, both the growth permissive and growth inhibitorycomponents both contain polyethylene glycol (PEG).

In some embodiments, the PEG is a multi-arm PEG.

In some embodiments, the PEG is comprised of a urethane or amidelinkage.

In some embodiments, the PEG comprised of an ester linkage.

In some embodiments, the PEG additionally comprises a linear end-cappedPEG of 5,000 Daltons or less.

In some embodiments, the crosslinking is performed between a PEG-NHSester and a PEG-amine or trilysine.

In some embodiments, the in situ forming gel contains pores 1 μm in sizeor larger.

In some embodiments, the in situ forming gel contains rods or filaments.

In some embodiments, the growth permissive component contains chitosan.

In some embodiments, the growth permissive component containspolylysine, preferably between 0.001 and 10 wt %, more preferablybetween 0.01 and 0.1 wt %.

In some embodiments, the nerve growth permissive components containsbetween 0.001 and 20% collagen, preferably between 3 and 6 w t %.

In some embodiments, the nerve growth permissive component containsbetween fibronectin.

In some embodiments, the growth permissive component containspoly-L-ornithine.

In some embodiments, the growth permissive component includes laminin,preferably between 0 and 5 wt %, more preferably between 0 and 0.5%.

In some embodiments, the swelling of the growth permissive component isless than 20%, preferably between 5 and 20%.

In some embodiments, the swelling of the growth inhibitory component isless than 30%, preferably between 0 and 10%.

In some embodiments, the swelling of the growth permissive component isless than or equal to the swelling of the growth inhibitory component.

In some embodiments, the compressive strength of the growth inhibitorycomponent is greater than 10 kPa, preferably >30 kPa.

In some embodiments, the growth permissive and growth inhibitorycomponent are different colors.

In some embodiments, the growth permissive region comprises agents thatsupport nerve survival, outgrowth, and regeneration.

In some embodiments, the growth permissive region permits infiltrationof Schwann or glial cells.

In some embodiments, a composition includes agents which may compriseone or more of growth factors, anti-inhibitory peptides or antibodies,and/or axon guidance cues.

In some embodiments, the system contains supporting cells such as glialcells, including Schwann cells, oligodendrocytes, or progenitor cellssuch as stems cells.

In some embodiments, the system is delivered to peripheral nerves or thespinal cord.

In some embodiments, the growth permissive and growth inhibitory regioninclude a P2XR receptor antagonist.

In some embodiments, the P2XR receptor antagonist is a P2X7 receptorantagonist, including Brilliant Blue FCF (BB FCF) or Brilliant Blue G(BBG).

In some embodiments, the P2XR antagonist is a P2X3 receptor antagonist,such as

In some embodiments, the concentration of the P2XR antagonist is between0.001 and 0.55% the hydrogel.

In some embodiments, disclosed is a kit including two or more in situforming hydrogels. The kit includes a dual applicator system clearlymarked with indicia as the growth permissive applicator and a dualapplicator system clearly marked as the growth inhibitory applicator.Each component can be clearly color coded and includes a powder vial, areconstitution/diluent solution, and an accelerator solution for use inthe dual applicator system. The kit also may include two or moreforms—one form for receiving the growth inhibitory hydrogel, the otherfor the growth permissive hydrogel.

In some embodiments, disclosed herein is a method of delivering dual insitu forming hydrogels to treat conditions involving nerves. The nervescan need repair, such as, for example, end-to-end anastomoses,coaptation, repair with allograft or autograft or conduit or wrap, orgap repair.

In some embodiments, a growth permissive region is delivered between theproximal and distal nerve stumps, between end-to-end anastomoses sites,between proximal stump and allograft/autograph.

In some embodiments, a growth permissive region is delivered between theproximal and graft and/or graft and distal stumps.

In some embodiments, a growth permissive region is delivered inside aconduit or wrap.

In some embodiments, a growth permissive region is delivered into a formthat permits adherence of the growth permissive gel to the nerves butnot the form.

In some embodiments, a growth inhibitory region is delivered after thegrowth permissive region.

In some embodiments, a growth inhibitory region covers the proximal anddistal nerves and growth permissive region.

In some embodiments, a kit can include an in situ forming hydrogel. Thekit includes a dual applicator system and a powder vial, areconstitution/diluent solution, and an accelerator solution for use inthe dual applicator system. The kit also may include a selection offorms in a range of sizes and lengths for receiving the hydrogel.

In some embodiments, a growth inhibitory region covers the anastomosesjunction.

In some embodiments, a growth inhibitory region covers the junction(s)between the nerve and the conduit or wrap.

In some embodiments, a growth inhibitory region covers a healthy,compressed, or contused nerve.

In some embodiments, disclosed herein is a formed in place nerveregeneration construct, comprising: a growth permissive hydrogel bridgehaving first and second ends and configured to span a space between twonerve ends and encourage nerve regrowth across the bridge; and a growthinhibiting hydrogel jacket encapsulating the growth permissive hydrogelbridge and configured to extend beyond the first and second ends todirectly contact the two nerve ends.

In some embodiments, disclosed herein is a method of encouraging nervegrowth between a first nerve end and a second nerve end, comprising:placing the first nerve end and the second nerve end in a form cavity;introducing a growth permissive media into the cavity and into contactwith the first nerve end and the second nerve end to form a junction;placing the junction into a second form cavity; and introducing a growthinhibiting media into the second form cavity to encapsulate thejunction.

In some embodiments, disclosed herein is a form for creating an in situnerve cap to inhibit neuroma formation, comprising: a concave walldefining a cavity, the wall having a top opening for accessing thecavity, the top opening lying on a first plane and having an area thatis less than the area of a second plane conforming to inside dimensionsof the cavity and spaced apart into the cavity and parallel to the firstplane; and a concave nerve guide carried by the wall and providing aside access to the cavity.

In some embodiments, disclosed herein is a form for creating an in situwrap around a nerve to nerve junction, comprising: a concave walldefining a cavity, the wall having a top opening for accessing thecavity, the top opening lying on a first plane and having an area thatis less than the area of a second plane conforming to inside dimensionsof the cavity and spaced apart into the cavity and parallel to the firstplane; a first concave nerve guide carried by the wall and providing afirst side access for positioning a first nerve end in the cavity; and asecond concave nerve guide carried by the wall and providing a secondside access for positioning a second nerve end in the cavity.

In some embodiments, disclosed herein is a composition for an in situforming growth inhibitory hydrogel with: compressive strength greaterthan 10 kDa for over 3 months, in vivo persistence for at least 3 monthscomprising less than 15% mass loss, and/or swelling of less than 30% forover 3 months.

In some embodiments, the composition includes one or more of:poly(ethylene glycol) succinimidyl carbonate, a P2XR receptorantagonist, and/or a P2X7 receptor antagonist.

In some embodiments, a P2X7 receptor antagonist is Brilliant Blue FCF(BB FCF) or Brilliant Blue G (BBG).

In some embodiments, a method of in situ formation of a nerve wrap,comprising identifying a section of a nerve; positioning the nerve in acavity defined by a form; introducing media into the cavity of the formto surround the nerve; and permitting the media to undergo atransformation from a first, relatively flowable state to a second,relatively non flowable state to form a protective barrier around thenerve.

In some embodiments, the nerve is healthy, compressed, or contused.

In some embodiments, the nerve is repaired through direct anastomoses,repair with allograft or autograft, or repair with a conduit.

In some embodiments, the method includes removing the form.

In some embodiments, the form comprises a nerve guide, and positioningcomprises positioning the nerve such that the nerve guide maintains thenerve spaced apart from a sidewall of the form.

In some embodiments, a method of in situ formation of a nerve wrapincludes where the nerve is covered circumferentially with at least 0.5mm of a protective barrier.

In some embodiments, the transformation occurs within about 10 secondsof the introducing step.

In some embodiments, the transformation comprises a crosslinking orpolymerizing.

In some embodiments, the transformation produces a synthetic crosslinkedhydrogel protective barrier.

In some embodiments, the protective barrier has an in vivo persistenceof at least about two months.

In some embodiments, the protective barrier has an in vivo persistenceof at least about three months.

In some embodiments, the transformation causes the media to swell involume within the range of from about 2% to about 60%.

In some embodiments, the transformation causes the media to swell involume within the range of from about 20% to 60%.

In some embodiments, the method includes forming a form in situ beforethe positioning the severed end; and/or delivering the media around thenerve in two successive steps.

In some embodiments, the severing a target nerve step and thepositioning a form at a treatment site step are accomplished by a singleinstrument.

In some embodiments, the viscosity of the flowable media is less than70,000 cps.

In some embodiments, the density of the flowable media is less than 1g/cm³.

In some embodiments, the form is comprised of silicone.

In some embodiments, the form contains an integral posts for seatinglonger lengths of the nerve.

In some embodiments, the wrap is comprised of PEG.

In some embodiments, the form has a clamshell lid.

In some embodiments, the growth permissive and growth inhibitory regioncontain a P2XR receptor antagonist.

In some embodiments, the P2X7 receptor antagonist is a P2X7 receptorantagonist, including Brilliant Blue FCF or Brilliant Blue G (BBG).

In some embodiments, the concentration of the P2XR antagonist is between0.001 to 0.55% in the hydrogel.

In some embodiments, disclosed herein are in situ forming hydrogel(s) asa cap. In some embodiments, the nerve cap is not pre-formed.

Some embodiments as disclosed herein include in situ forming hydrogelscaffolds or ones that can be formed/wrapped in situ around a nerve. Insome embodiments, systems and methods do not include a nerve guidanceconduit (tube with two open ends) rather than a cap. In someembodiments, disclosed herein are systems and methods for delivering thehydrogel circumferentially around the nerve in appropriately designedforms. In some embodiments, systems and methods can include use of aform or the specific design of the PEG hydrogel to prevent neuromaformation such as circumferential delivery, in vivo persistence, minimalswelling etc. and delivering them into a removable form.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a perspective schematic view of a nerve end positioned withina form cavity. An entrance region that permits the nerve to guided intothe form. The length of the form is provided to provide a sufficientsurface area over which the hydrogel to form and adhere to the nervetissue.

FIG. 1B is a side elevational cross section through the construct ofFIG. 1A.

FIG. 1C is a top view of the construct of FIG. 1A.

FIG. 1D is an end view of the construct of FIG. 1A.

FIG. 1E is a cross-sectional view taken along the line 1E-1E in FIG. 1B.

FIG. 2 is a schematic illustration of a formed barrier formed inaccordance with some embodiments of the present invention.

FIG. 3 is a perspective view of a form, having a stabilizing feature.

FIG. 4 is a perspective view of a form for creating a wrap around anerve or a growth permissive region between a nerve. Thus, depending onthe application, the wrap form may contain a growth permissive or growthinhibitory hydrogel.

FIGS. 5A-5E illustrate a series of steps for creating a growthpermissive hydrogel junction encapsulated by a growth inhibitoryhydrogel barrier.

FIG. 6 is a perspective view of a clamshell form.

FIGS. 7-10C illustrate embodiments of tools for transecting nervesand/or creating a hydrogel junction.

FIGS. 11A-11E illustrate views of a form and methods of use.

FIG. 12 is a perspective view of a form with a stabilizing rod.

FIGS. 13A-13D is a perspective view of a cap form with a partial coverand in internal rod to support the nerve.

FIGS. 14A-14C is a perspective view of a cap form with a partialclamshell.

FIGS. 15A-15C is a perspective view of a tearable nerve cap form.

FIGS. 16Ai-16E illustrate perspective views and photographs of in situformed hydrogels (growth inhibitory and growth permissive) around nervesboth in cap and wrap form.

FIGS. 17A-17B illustrate preclinical data demonstrating the formation ofneuroma after the delivery of hydrogels with adequate initial mechanicalstrength but inadequate in vivo persistence relative to hydrogels withlonger duration mechanical strength and persistence.

FIGS. 18A-18B illustrate a mixing element design to improve theconsistence of the hydrogel when delivering low volumes of precursorsolution.

DETAILED DESCRIPTION

Some aspects of the present invention involve in situ formation of aprotective barrier around an end of a nerve using injectable orsurgically introduced media which may be a gel/hydrogel or gelprecursors to block nerve regeneration and/or neuroma formation andinflammation and adhesion etc. around/in contact with nerves. Access maybe by way of an open surgical approach or percutaneous (needle,endovascular/transvascular). The nerve end or stump may be formed bytransection (cutting), traumatic injury, or ablation through any of avariety of modalities including RF, cryo, ultrasound, chemical, thermal,microwave or others known in the art.

The hydrogels may ‘adhere’ to the end of the nerves providing a snug,conforming, cushioning barrier around the end of a nerve as opposed to acap with a void (inflammatory cells/fluid cysts present causing neuromaformation). Hydrogels are transparent for visualization, low-swelling,compliant, and are delivered into a form to generate hydrogel caps withvolumes 0.1 to 0.5 ml. The barrier may inhibit neuroma formation purelythrough mechanical blocking of nerve regrowth. The media mayadditionally comprise any of a variety of drugs such as for inhibitingnerve regrowth as is discussed in further detail herein.

Target nerves can vary widely in diameter or non circular outsideconfiguration, and the cut or severance angle and precision can alsovary. In accordance with some embodiments of the present invention,capping is best accomplished by forming a soft, cushioning andconformable protective barrier in situ. A flowable media or mediaprecursor(s) may be introduced to surround and conform to theconfiguration of the nerve end, and then be transformed into a nonflowable state to form a protective plug in close conformity with andbonded to the nerve end. To contain the media before and duringtransformation (e.g., crosslinking), the media may be introduced into aform into which the nerve end has previously or will be placed. Fillingthe media into a form allows the media to surround the nerve end andtransform to a solid state while contained in a predetermined volume andconfiguration to consistently produce a protective, conforming nerve capregardless of the diameter and configuration of the nerve stump.

Referring to FIGS. 1A through 1D, there is illustrated a nerve cap form10. The form 10 extends between a proximal end 12, a distal end 14 andincludes a side wall 16 extending there between. Sidewall 16 is concaveto produce a form cavity 18 therein. The form cavity 18 is exposed tothe outside of the form by way of a window 20.

The proximal end 12 of the form 10 is provided with a nerve guide 22 tofacilitate passage of the nerve 24 to position the nerve end 26 withinthe form cavity 18. The nerve guide 22 may comprise a window or openingin the proximal end wall 12 of the form 10, and is configured to supportthe nerve at a level that positions the nerve end 26 within the formcavity 18. In the illustrated embodiment, the nerve guide 22 includes asupport surface 28 on an upwardly concave housing to produce a nerveguide channel 30. See FIG. 1D. FIG. 1E is a cross-sectional view takenalong the line 1E-1E in FIG. 1B.

Referring to FIGS. 1B and 1C, the nerve end 26 is positioned such thatat least 1 mm and preferably 2 mm or more in any direction separate thenerve end 26 from the interior surface of the side wall of the form 10.This permits the media 27 to flow into the form cavity and surround thenerve end 26 to provide a protective barrier in all directions.

Following transformation of the media from a relatively flowable stateto a relatively non-flowable state, the form 10 may be left in place, ormay be peeled away to leave behind a formed barrier 60 in the form of aplug as is schematically illustrated in FIG. 2.

In order to stabilize the form 10 following placement and during thefilling and transformation stages, at least one stabilizing feature 32may be added. See FIG. 3. The stabilizing feature 32 maybe at least oneor two or four or more ridges, flanges or feet which provide atransverse support surface 34 for contacting adjacent tissue andstabilizing the form against motion. The transverse support surface 34may extend along or be parallel to a tangent to the sidewall of the form10.

In one implementation of the invention there is provided a dual hydrogelconstruct with connectivity across the junction between two nerve endsachieved by creating a growth permissive hydrogel junction between thetwo opposing nerve ends, then encapsulating that junction with a growthinhibitory hydrogel capsule. The use of an in situ crosslinking hydrogelfor the growth permissive media produces a junction with sufficientmechanical integrity and adhesiveness that it can be picked up as a unitas if it were an intact nerve and then placed in a second form to formthe outer growth inhibitory hydrogel capsule.

Referring to FIG. 4, a form 10 includes a curved side wall 66 defining aform cavity 68. A first nerve guide 70 and a second nerve guide 72 arein communication with the cavity 68 and dimensioned and oriented toallow positioning first and second nerve ends into the cavity 68 in aposition where they will face each other and become surrounded byflowable media introduced into the cavity 68.

Referring to FIGS. 5A-5E, there is illustrated a sequence of steps forforming a dual hydrogel conductive nerve junction between two nerveends. A first form 50 comprises an elongate side wall curved to form aconcavity such as in the form of a half of a cylinder, having an insidediameter larger than the diameter of a target nerve. The form 50 has afirst end 52, a second end 54 and an elongate channel 56 extending therebetween. The first nerve end 58 is positioned within the channel 56 fromthe first end 52. A second nerve end 60 extends into the channel 56 fromthe second end 54. The result is a form cavity 62 formed between thefirst and second nerve endings and the sidewall of the form 50.

A transformable growth permissive hydrogel precursor is introduced intothe form cavity 62 to adhere to the nerve ends and polymerize in situ toform a conductive bridge 64 between the first nerve end 58 and secondnerve end 60 as shown in FIG. 5B. Following transformation of the gel toa less flowable state, the form 50 is removed as shown leaving ajunction comprising the nerve ends connected by a conductive bridge 64of polymerized growth permissive gel 62. See FIG. 5C.

Thereafter the polymerized junction is placed within a second form 66having a central chamber 68 separating first and second nerve supports70, 72, such as that illustrated in FIG. 4. A second growth inhibitoryhydrogel precursor is introduced into the central chamber 68 to surroundand over form the conductive bridge 64 and nerve ends to produce a finalconstruct in which the first growth permissive polymer bridge 62 isencapsulated by second, growth inhibitory polymer capsule 70. See FIG.5E.

Either the nerve capping or nerve regeneration forms of some embodimentsof the present invention can be provided in a clam shell configurationsuch as that illustrated in FIG. 6. A first shell 80 defines a firstcavity 82 and a second shell 84 defines a second, complementary cavity86. The first and second shells are joined by a hinge 88 such as aflexible living hinge made from a thin polymeric membrane. The first andsecond shells 80, 84 can be rotated towards each other about the hinge88 to form an enclosed chambered form.

FIG. 7 illustrates a perspective view of a clamping tool 700 configuredto cut nerve tissue, as well as to house a form for forming a hydrogelnerve junction such as disclosed elsewhere herein, e.g., aftertransecting a nerve. The tool 700 can include a plurality of proximalmovable grips 702, each connected to shafts 706 connected at pivot 704,and can have an unlocked configuration as shown, movable to a lockedconfiguration utilizing a locking mechanism 705, such as a series ofinterlocking teeth. The distal ends 707 of the shafts 706 can includeend effectors 708 that can include sidewalls 710 that can have a curvedgeometry as shown, and complementary cutting elements 712 operablyconnected to the curved sidewalls. In some embodiments, a form 10 can beconnected to the sidewall 710 after cutting the nerve. In otherembodiments, a form can include an integrally formed cutting element. Insome embodiments, the cutting element can be detached or otherwiseremoved after cutting, leaving the form in place.

FIG. 8 is a close-up view of an end effector 708 of FIG. 7, alsoillustrating that the end effector 708 can also carry a form 10. FIG. 9is a side close-up view of the distal end of an embodiment of the tool,illustrating that each of the end effectors can include cutting elementsand/or forms.

FIGS. 10A-10C illustrate various stages of a method of transecting anerve while removing axoplasm from the nerve tip, to improve closeapposition between the nerve end and the hydrogel. In some embodiments,opposing end effectors 708 can include blades 712, which can be of equalor unequal length. Blades 712 on each end effector 708 can be generallyopposing, but offset from each other as shown in some embodiments.Actuating the end effectors 708 can result in the blades transecting thenerve 24 creating a nerve end 26. The blades can be within a form aspreviously described. An absorbent material 780 such as a swab can beconnected to one or more end effectors 708 (such as within a form, forexample) and be proximate, such as directly adjacent one or more of theblades 712, in order to absorb any axoplasm after nerve transection. Thetip of the swab can be, for example, less than 5 mm, more preferablyless than 2 mm in order that it may fit comfortably within the form andhold the nerve while the hydrogel is delivered.

Referring to FIGS. 11A-11E, in some embodiments, a delivery needle 1102is advanced into an opening 1104 of the cap form 1100 to deliver thehydrogel precursor in and around the nerve 1124. Also shown is nerveguide 1122 which can be as described elsewhere herein. See FIG. 11A.Hydrogel may be delivered in to successive applications, to half fillthe form and for a hydrogel 1150 as shown in FIG. 11B and thencompletely fill the form as shown in FIG. 11C and form a hydrogel capafter which the form is removed. Hydrogel is delivered in a small bolus1152 to surround the tip of the nerve as shown in FIG. 11D and then theremainder of the cap is subsequently filled to form a hydrogel cap afterwhich the form is removed as shown in FIG. 11E.

Referring to FIG. 12, in some embodiments, a biodegradable rod 1215 isplaced adjacent to and in continuity with the length of the nerve 1224.The rod 1215 provides additional strength to the nerve 1224 andnaturally adheres to the nerve 1224 such that, irrespective of the rod'sposition, the nerve 1224 adheres to the rod 1215. The hydrogel solutionis then delivered on or around the nerve 1224 and the biodegradable rod1215 to form a nerve cap.

Referring to FIGS. 13A-13D, in some embodiments, one, two, or moreapertures 1310 are provided in the side of a cap or wrap form 1300 toguide the needle to deliver the precursor solution in the correctlocation. The hole 1310 may be in one of many locations around the formas is needed to deliver the precursor solution 13A. A post 1330 may beincluded in the bottom of a cap or wrap form to provide additionalsupport to the nerve. The nerve length is rested on top of the post 1330while taking care that the tip of the nerve does not come in contactwith the post 1330. The post 1330 may be integral to the cap or wrapform and is subsequently removed when the form is removed.Alternatively, the post 1330 may comprise a biodegradable post thatremains integral to the hydrogel cap. See FIGS. 13B and 13C. In someembodiments, a cap form can include a partial lid 1320, shown in FIG.13A. The form is tilted such that the precursor material will flow toand fill the distal cap first, surrounding the proximal nerve stump endand then subsequently fill the rest of the nerve cap. As shown in FIG.13D, the cap or wrap form can also include raised tabs 1333.

FIGS. 14A-14C illustrates various views of an embodiment of a nerve capform 1400 similar to that shown in FIGS. 13A-13D with a partial lid 1420connected via a hinge 1428 with an insert 1440 to assist in centeringthe lid 1420 on the cap form 1400. Also shown is nerve guide, which canbe as described elsewhere herein.

FIGS. 15A-15C illustrate various views of a tearable cap form 1500 thatcan include a peelable sheath 1560 including a sidewall 1561, into whichthe nerve is placed (nerve channel 1562). The precursor solution isdelivered into and around a first nerve channel 1562 and the peelablesheeth 1560 is subsequently torn off the nerve 1524, such as using atearable tab 1564 as shown in FIG. 15A. The nerve hydrogel 1570 is thenrotated approximately 90 degrees and placed in a second larger diameterpeelable cap form 1501. The precursor solution is then applied into thenerve channel to surround the nerve and the first cap form. The peelablesheath is then torn off the second tearable cap form 1501. The resultantcylindrical cap form contains the centered nerve. The nerve 1524 canthen be rotated back to the normal physiologic position, as shown inFIG. 15B. FIG. 15C illustrates an alternate tearable cap form designwhich can include a plurality of tabs.

FIG. 16Ai-16E illustrate hydrogel filling and surrounding a nerve in acap form. FIG. 16B illustrates a photograph of the hydrogel formedinside a cap form. FIG. 16C illustrates a high resolution image of a capform. FIG. 16D illustrates an example of a cap form and wrap formsaround the pig sciatic nerve. Example of a growth permissive hydrogel(pink) in a wrap form around a nerve and then subsequently embedded in asecond (blue) growth inhibitory hydrogel wrap. Hydrogels are cut incross section in order to see the growth permissive (pink) hydrogelembedded within the growth inhibitory (blue) hydrogel, as shown in FIG.16E.

FIG. 17A illustrates neuroma formation after delivery of DuraSeal in acap form around a transected rat sciatic nerve. FIG. 17B illustrates theabsence of neuroma formation after delivery of the lead formulationaround a transected rat sciatic nerve. Cap form maintains mechanicalstrength and in vivo persistence of 3 months.

FIGS. 18A-18B schematically illustrates an embodiment of a mixingelement to mix a two-part hydrogel system. In some embodiments, onestatic mixer 1800 delivers the hydrogel precursor solution into acentral chamber, permitting the backflow and recirculation of theinitial material coming out of the mixer. A second static mixer capturesthe well mixed solution and delivers it through the needle tip. Thefluid entrance 1802 (from a dual chamber applicator) and fluid exit 1804(to a blunt needle) are also shown.

The table to follow is related to specific non-limiting embodiments anddevices for delivering in situ forming hydrogels.

Delivery of in situ forming Hydrogels to: Form Shape Selected Hydrogel 1) Undamaged nerves Wrap Growth Inhibitory 2) Nerves that have beencompressed, Wrap Growth Inhibitory contused or stretched 3) Stumpneuroma or transected nerve that Cap Growth Inhibitory can not berepaired 4) Nerves that have been transected and Wrap Growth Permissiveundergone direct suture repair and then Growth (coaptation, end-to-endanastamosed) Inhibitory 5) Nerves that have undergone suture repairWrap(s) - protect Growth Permissive and placement in a nerve conduit orwrap nerve-conduit and then Growth junction Inhibitory 6) Nerves thathave undergone connector Wrap(s) - protect Growth Permissive assisted‘suturelesss’ neurorraphy in nerve-conduit juncton and then Growth whichsutures are placed between the Inhibitory epineurium and the connectorbut not to one another 7) Nerves that have been placed in a Wrap(s) -protect Growth Permissive connector without suturing anastamoses andthen Growth Inhibitory 8) Nerves that are undergoing conduit Wrap(s)protect Growth Permissive detensioning gap repair nerve-conduit and thenGrowth junction Inhibitory 9) Nerves undergoing detensioning allograftWraps(s) - protect Growth Permissive interposition with connectorassisted nerve-nerve and then Growth sutureless repair anastamosesInhibitory 10)  Nerves undergoing detensioning Wrap(s) - GrowthPermissive autologous nerve graft interposition suture protect nerve-nerve and then Growth repair Inhibitory 11)  Nerves that have non-uniongaps (e.g. 2 Wraps Growth Permissive cannot be repaired directly) andthen Growth Inhibitory 12)  Nerves that have been repaired and one orWrap(s) - Protect Growth Permissive more wraps are placed around thenerve-wrap and then Growth anastomoses site Inhibitory 13)  Nervesundergoing suture repair in Wrap - Protect Growth Permissive targetedmuscle reinnervation nerve-nerve interface and then Growth Inhibitory

Peripheral Nerve Stimulation (PNS). As neurostimulators have advancedfrom the spine to the periphery and the hardware has been miniaturized,purpose built peripheral nerve stimulators are being developed andadvanced for blocking pain, stimulating muscle contractions, andstimulating or blocking nerves to modulate disease and/orsymptomatology, and stimulate nerve regeneration. As new applicationsand new neurostimulators have been developed, so has an increasedawareness of the need to be able to maintain the stimulation electrodesand catheters in direct or close apposition with the target nerve as 1)placing the electrodes next to the nerve procedurally can be challengingand electrodes can migrate procedurally even after ideal placementadjacent to a nerve and 2) after placement, electrodes may drift throughpatient movement or handling as muscles contract or the implant getsbetter seated within the tissue. This can lead to loss of the therapyreaching the target nerve and thus loss of efficacy.

Percutaneous delivery. With the advent of higher resolution handheldultrasound and better training amongst interventional pain physicians,percutaneously delivered implantable neurostimulators are increasinglybeing used as an alternate method to treat chronic pain. In oneembodiment once an electrode has been placed adjacent to a nerve using apercutaneous delivery system, the position of the electrode next to thenerve can be maintained by delivering approximately 0.1 to 3 cc of anelectroconductive hydrogel to form around the electrode and maintain itin close apposition with the nerve. The hydrogel media can be deliveredthrough the lumen of the catheter delivery system or the lumen of theelectrode and will form in situ. In some embodiments, the surface of theelectrode can be designed such that it interfaces is rougher, permittingstronger intercalation between the hydrogel and the electrode to preventlead migration. In other embodiments, a coil or other screw like designis placed on the end of the electrode to provide better purchase betweenthe electrode, the hydrogel, and the surrounding tissue. For thepercutaneous applications, delivery of a growth inhibitory hydrogels orhydrogels with medium to long duration mechanical strength aredesirable. Again, longer-term the maintenance of mechanical strength tomaintain the position of the electrode within the hydrogel is desirableuntil the chronic foreign body response is sufficient to hold theelectrode in place. For example, to maintain longer term lead placement,the selection of crosslinked PEG hydrogels containing more stable ester,urethane or amide linkages is desirable, such as PEG-SG, PEG-SC, orPEG-SGA.

In still other embodiments, the neurostimulators are injectable wirelessimplants and takes the form of a pellet, rods, beads, a wrap a sheet ora cuff that are held in place with a hydrogel adjacent to a nerve,ganglia, or plexus. In one embodiment, the hydrogel is delivered firstto the target site and the neurostimulator is delivered into thehydrogel. In another embodiment, the neurostimulator implant(s) isdelivered first, adjusted to the desired location and then the hydrogelis then delivered around it to secure it in the desired location.Similarly, the neurostimulator implant location may be adjusted using anexternal magnet to orient the implant adjacent to or in contact with thenerve or neural tissue. In this embodiment, the gelation time can beadjusted to provide sufficient time for the appropriate alignment of theneurostimulator. In some embodiments, a plurality of injectablemicrostimulator implants are injected into a degradable or nondegradable in situ forming hydrogel. In yet another embodiment,microstimulators in the form of micro or nanorods are implanted in thegrowth permissive hydrogel between the two nerve stumps to promoteneurite extension and accelerated regeneration. These microstimulatorsmay deliver magnetic, chemical, or electric fields to stimulate nerveregeneration through the gel and potentially along the microstimulatorimplants. In one embodiment, the microstimulators are nanofibers and canbe injected through a low gauge needle or catheter to the nerve. Inanother embodiment, short- or long-acting microstimulators can bedelivered with an injectable biocompatible biomaterial such as ahydrogel to form a neurostimulator anisogel. The microstimulators aremagnetic, allowing directional control of the microstimulator implantand, for example, parallel alignment of the microimplants within thehydrogel prior to the gel converting from a precursor solution to a gel.These hydrogels would be injected around or in proximity to nervebundles or tendrils and then the microstimulators may physically provideregions across which they can grow to and orient along as well asproviding chemical, electrical, or magnetic field stimulation to supportneurite outgrowth.

Open surgical. For open surgical applications, the hydrogel may also bedeposited in a similar manner around the electrode with the electrode indirect contact and/or adjacent to the nerve under direct visualization.Again, deposition of approximately 0.5 to 1 cc of hydrogel is sufficientto maintain the electrode position relative to a nerve. The electrodecan be inserted into a groove in the silicone form adjacent to and withthe nerve prior to the delivery of the hydrogel. Forms can be envisionedthat have a second entrance region for the electrode. In this manner,for example, the electrode can be aligned to run parallel to the nerveor in direct apposition to the nerve when the gel is applied. Forapplications where the neurostimulation therapy is only required for aday to several weeks, pulling on the electrode will cause it to beremoved from the hydrogel with relative ease. Utilizing combinations ofgrowth inhibitory and growth permissive hydrogels described above, maybe selected depending on the application. For examples in whichelectrodes placed next to the nerve only need to stay in place for amatter of days or weeks, a shorter term degradable hydrogel may beemployed. This provides sufficient time for the hydrogel to remain inplace while the therapy is delivered and then be rapidly cleared fromthe tissue. One example of this would be the selection of crosslinkedPEG hydrogels containing more reactive ester linkages such as PEG-SS orPEG-SAZ. These hydrogels are electrically conductive and thus suitablefor applications involving neurostimulators.

Generally, the selection of low swelling formulation is critical tomaintain apposition with the electrode; in one embodiment, the hydrogelswelling is less than 30%, more preferably less than 20% in order tomaintain apposition with the nerve and the electrode.

In yet another embodiment, the in situ forming hydrogel can be used tosecure a convection enhanced delivery system to the site. Like theimplantable neurostimulator, a drug delivery catheter can be securedapproximately 10 mm proximal to an injury nerve site with the tipapproximately 5 mm from the nerve injury. Like the implantableneurostimulator, the silicone form can be designed to include anentrance zone or cut out of the top edge of the silicone form to permitthe catheter or stimulator lead to rest in the form in preparation foraddition of the hydrogel. After delivery of therapy (neurostimulation,convection enhanced drug delivery), the catheter or neurostimulator canbe removed from the hydrogel without disrupting the protective barrieraround the hydrogel. For example U.S. Pat. No. 9,386,990 teaches the useof DuraSeal to repair nerves with an in vivo persistence of two to fourweeks, the hydrogel does not provide the sustained mechanical strengthnecessary to prevent neuroma formation or detension a nerve duringregeneration, such as at 3 and 4 months after surgical repair. Forexample, crosslinked multi-arm PEGs containing rapidly degrading esterlinkages such as PEG-SS or PEG-SG are well-suited suitable forapplications to prevent the acute and subacute adhesion formation. Foranother example, low molecular weight linear PEGs have been demonstratedto act as a fusogen and promote nerve repair and regeneration wheninjected around injured nerves (but do not provide mechanical strengthor persistence to prevent neuroma formation. For example, PEG hydrogels,such as PEG tetraacrylate hydrogels, have been used to rejoin nerves inpreclinical models (Hubbell 2004/0195710)

None of the previous examples contain the degradable linkages necessaryto support the required mechanical strength or in vivo persistencerequired for applications to prevent aberrant nerve outgrowth andneuroma formation. Commercially available PEG hydrogels, particularlyconventional PEGs with a hydrolytic ester linkage, do not have thesuitable mechanical strength or in vivo persistence to prevent neuromaformation or detension a nerve for three of four months until the nerveis repaired. These PEGs and PEG gels may have sufficient mechanicalstrength initially to temporarily assist in the repair of nerves acrossan anastomoses and/or prevent adhesion formation, but the hydrogels donot have sufficient mechanical strength at two months, or morepreferably three months post administration to prevent aberrant neuromaformation (cap) and continue to provide mechanical offloading to supportto a regenerating nerve (wrap). FIG. 16 provides an example of the lackof durability of the DuraSeal hydrogel in preventing neuroma formationin a rat sciatic nerve transection model. The hydrogels containing esterlinkages have either degraded sufficient that they no longer provide abarrier to nerve regeneration, have fallen off the nerve, or have beencleared entirely. As a result, the initial mechanical barrier was notsufficient to act as a long-term barrier to prevent nerve outgrowth andneuroma formation.

Furthermore, the other approaches teach delivering the an in situforming hydrogel around the nerves directly without protecting theunderlying muscle from adhesions or providing a method to systematicallycircumferentially cover the proximal nerve tip with hydrogel. In situforming polymeric systems adhere to, albeit with varying to degrees, tothe surrounding tissues that they come into contact with duringcrosslinking or polymerization. If the non-target tissue (e.g. muscle orfascia) is not protected or shielded from the reaction, the hydrogelalso adheres to this tissue. Since nerves glide freely within a fascialplane, typically between muscles, limitation to their movement is notdesirable and may result in pain and or loss of efficacy. Someembodiments described here provide forms that separate the in situforming hydrogel from the surrounding environment, preventing tetheringbetween the nerve and the surrounding tissue and permitting the nerve toglide within the fascial tunnel.

Nerve blocking. In order to block nerve regeneration, the in situforming biomaterial needs to have the physical properties to preventnerves from migrating into the biomaterial including negative or neutralcharge, smaller pore size, hydrophillicity and/or higher crosslinkingdensity. Although most studies are focused on materials through whichnerves regenerate, several studies have documented the biomaterialsthrough which nerves will not grow, including poly(ethyleneglycol)-based hydrogels, agarose- and alginate-based hydrogels,particularly at higher concentrations of the polymers. Higherconcentrations typically have higher crosslinking density and thussmaller pore size. These hydrogels can be employed for their ability toprevent neurite outgrowth in vitro and in vivo by virtue of theircharge, inert surface, hydrophilicity, and pore size. One example ofthis agarose, in which nerves will not extend across the biomaterialabove concentrations of 1.25% wt/vol. In another example, PEG hydrogelscan prevent neuroma formation at 4% w/v and higher. In otherembodiments, even positively charged or natural in situ formingbiomaterials can provide a barrier to nerve regeneration if the solidcontent and crosslinking density are such that the pores are too smallfor cellular ingrowth.

In order to prevent neuroma formation, the in situ forming biomaterialneeds to provide the requisite mechanical strength to act as a barrierto nerve regeneration for two months, more preferably three months ormore. Many in situ forming gels, including commercially in situ formingPEG hydrogels with biodegradable ester linkages, may have sufficientmechanical strength initially but hydrolyze at such a rate that theircrosslinks have broken sufficient that their mechanical strength at 1 to2 months is not sufficient to prevent neuroma formation (See Table 1).In vivo experiments in a rat sciatic nerve model demonstrated theformation of bulbous neuromas at between one and three months afterdelivery of these hydrogels around the end of a transected nerve stump.Preclinical testing has demonstrated that a mechanical strength of atleast 10 kPa, more preferably 20 kPa or more is necessary to preventneuroma formation. At three months, in vivo studies have demonstratedthat these hydrogels have been full degraded and cleared from the siteor have lost their mechanical integrity sufficient that the nerve hasgrown out into the soft, collapsed and/or fractured gels and formed aneuroma. Thus, although the prior art teaches the use of PEG hydrogelsfor the purposes of nerve repair, not all hydrogels are suitable tosupport the long-term mechanical strength and persistence requirementsnecessitated to prevent neuroma formation and aberrant nerve outgrowth.Preferably the barrier has an in vivo persistence of at least about twomonth or at least about three months, preferably four months or moredepending upon the desired clinical response to reduce chronicneuropathic pain after surgery. The mechanical integrity of thehydrogels at various points in vitro and in vivo can be assessed throughcompression testing, described further below.

Persistence The in vivo persistence of biodegradable hydrogels isrelated to the crosslinking density and thus the mechanical integrity ofthe hydrogel. For applications to prevent neuroma formation, thehydrogel degradation must be sufficiently slow that the hydrogel doesnot lose significant structural integrity during the weeks to monthsduring which the nerves are attempting to regenerate, which occurs overapproximately 3 months and may be 6 months or more in humans. In thismanner, the persistence of the hydrogel and the persistence of themechanical integrity of the hydrogel is critical to providing ongoingprotection and padding from neuroma and aberrant nerve outgrowthpreferably for 3 months or more, preferably 4 months or more. Inembodiments utilizing a degradable hydrogel, the mechanical strengthmust be maintained for longer than 2 months, preferably 3 months andthus there must be no substantial degradation of the hydrogel for thisperiod of time, preferably 3 months or more. Similarly, the persistenceof the mechanical integrity and, in turn, the hydrogel is critical tothe ongoing offloading provided by the hydrogel around the nerve-nerveor nerve-graft interface for a period of preferably 2 months, morepreferably 3 months as even nerves that have been directly sutured toone another through direct coaptation still have not regained theiroriginal strength (nerves have approximately 60% of original strength at3 months after a transection).

The development of in situ forming polymers, and particularly, in situforming synthetic hydrogels, including PEG-based hydrogels with longerin vivo mechanical strength and longer persistence profiles beyond 2.5months but less than 12 months is challenging. For example, there is asignificant gap between the in vivo persistence of PEG hydrogels withbiodegradable esters (weeks to less than 3 months) in and around thesurgical environment of the nerve and PEG hydrogels containingbiodegradable urethane or amide bonds, with degradation profiles in thissubcutaneous extramuscular location on the order of 9 months to 18months or more. Some embodiments focus on in situ forming polymers,preferably multi-arm PEGs, with the requisite mechanical strength andpersistence to prevent neuroma formation. In particular, the swelling,mechanical strength and in vivo persistence of PEG hydrogels aredescribed to permit the long-term safety and efficacy in applicationsrequiring the long-term prevention of aberrant nerve outgrowth and theability to detension and offload nerves over a period of months afterthe surgical repair.

In order to obtain a suitable in vivo mechanical strength andpersistence, conventional PEG hydrogels containing a degradable esterlinkers that are widely available commercially as dural and lungsealants are not suitable for applications around nerves given theirloss of mechanical strength and/or clearance within a couple months.Simply, degradation occurs at a rapid enough rate that mechanicalintegrity can not be maintained for sufficient time, making thesehydrogels suitable for anti-adhesion prevention but not the preventionof nerve outgrowth. In embodiments utilizing a nondegradable PEGhydrogel, the mechanical strength of the hydrogel is based on theinitial mechanical strength of the hydrogel as the crosslinks do notdegrade over time. In vitro and in vivo testing of a range of hydrogelwith various molecular weights, degradable linkages, crosslinkingdensities demonstrated that only hydrogels with sufficient mechanicalstrength at 3 months (and with this, in vivo persistence) were able toprevent neuroma formation. Examples of hydrogels, degradation times, andformation of neuromas are provided in the table below. FIGS. 16Ai-16Aiiiillustrate the formation of a neuroma after the delivery of DuraSeal.

Examples of Multi-Armed PEG Hydrogels with Various Hydrolytically labilebonds

Neuroma formation in Rat Sciatic PEG Hydrogel In Vivo Persistence NerveTransection Model PEG-SS (ester bond) 2 weeks Large bulbous neuromaobserved at 1 month Duraseal (ester bond) 2 to 8 weeks Large bulbousneuroma formation observed at 2 and 3 months PEG-SG (ester bond) 4 to 8weeks Large bulbous neuroma formation observed at 3 months PEG-SAP(ester bond) 6 to 8 weeks Large bulbous neuroma formation observed at 2and 3 months PEG-SAZ (ester bond) 2-3 weeks Large bulbous neuroma at 2and 3 months PEG-SGA (amide bond) 9 months or more No neuroma formationPEG-SC (urethane bond) 6 months or more No neuroma formation

In vivo persistence refers to the absence of significant absorption ofthe biomaterial, such as less than 25% resorption, preferably less than15% at a given time point. Depending on the biomaterial, this can beassessed by mass loss, loss of cross-linking density, or change in theform of the biomaterial. Active bonds that have more extendeddegradation in vivo such as the PEG-ureas (e.g. PEG isocyanate,PEG-NCO), PEG-urethanes (PEG-succinimidyl carbonate) (PEG-SC) andPEG-carbamate. Hydrogels comprised of polyethylene glycol succinimidylcarbonates (PEG-SCs) with more than 2 arms, such as the 4-arm, 6-arm, or8-arm PEGs with molecular weights ranging from 1K to 50K, preferably 10Kto 20K, such as 10K, 15K or 20 kDa. In some embodiments, the 4-arm 10KPEG-SC, 4-arm 20K PEG-SC, 8-arm 10K PEG-SC, 8-arm 15K PEG-SC, or 8-arm20K PEG-SC are selected, more preferably 4-arm 10K PEG-SC or 8-arm 20KPEG-SC. The following patent is incorporated for reference20160331738A1.

Compressive Strength.

The desired compressive strength (elastic modulus, Young's modulus) ofthe hydrogel is greater than 10 kPa, preferably greater than 20 kPA,preferably greater than 30 kPa. In the preferred embodiment, thecompressive strength of the is greater than 10 kPa after 3 months invivo, more preferably 40 kPa at 3 months after administration.

Compressive strength was measured benchtop after in vitro equilibriumand also after harvesting implanted samples from the subcutaneous spacein rats, in which hydrogel cyclinders (d=6 mm) are cut to 100 mm long,pre-equilibrated (for 12 hours at 37° C.) and evaluated for compressivestrength. Compressive properties of the hydrogel formulations weremeasured at a 1 mm/min with the Instron. The modulus was calculated asthe tangent slope of the linear region between 0.05 and 0.17 of thestress-strain curve.

Compressive Strength of Various Formulations

Compressive Compressive modulus Neuroma Polymer modulus (t = 0) (t = 3months, in vivo) formation Formulation G 20 kPa 5 kPa Neuroma formationFormulation H 12 kPa 8 kPa Neuroma formation Formulation I 1 kPa 1 kPaNeuroma formation Formulation I 25 kPa 17 kPa No neuroma formationFormulation J 72 kPa 55 kPa No neuroma formation

Although in vitro mechanical strength and persistence of the hydrogels(37° C., PBS) typically does not correlate well with in vivopersistence, the maintenance of mechanical strength of the hydrogels at3 months in vitro is a strong indicator of the ability of the hydrogelto provide a sustained mechanical barrier to nerve regeneration in vivo.

In some embodiments a cleavable carbamate, carbonate, or amide linker ina biodegradable hydrogel permits a more stable slowly degrading bond tomaintain the requisite mechanical strength to prevent nerve outgrowthfor three months or more and, with this, the in vivo persistence toprovide the sustained mechanical barrier to nerve regeneration.

Generally, the structure of multi-armed PEGs are

C-[(PEG)_(n)-M-L-F]_(m)

where

C=core structure of the multi-arm PEG

n=repeating units of PEG on each arm (25 to 60 units)

M=Modifier

L=cleavable or noncleavable linker (ester, urethane, amide, urea,carbamate, carbonate, thiourea, thioester, disulfide, hydrazone, oxime,imine, amidine, triazole and thiol/maleimide).

F=reactive functional group for covalent crosslinking, e.g. maleimide,thiol or protected thiol, alcohols, acrylates, acrylamides, amines,protected amines, carboxylic acids or protected carboxylic acids,azides, alkynes, 1,3-dienes, furans, alpha-halocarbonyls, andN-hydroxysuccinimidyl, N-hydroxysulfosuccinimidyl, or nitrophenyl estersor carbonates

m=number of PEG arms (e.g. 2, 3, 4, 6, 8, 10)

In some embodiments, hydrolysis modifiers (M) can be incorporated intothe backbone of the hydrogels to slow the hydrolytic degradation of theester bonds (L) in the hydrogel. This can be accomplished with electrondonating groups which regard the reaction or by increasing the length ofthe carbon chain adjacent to the ester bond in order to increase thehydrophobicity and shield the bond from hydrolysis. For example,PEG-SAP, PEG-SAZ are examples of PEG-ester bonds with longer carbonchains than PEG-SG. In another embodiment, an aromatic group is placednext to the ester group to provide additional stability of the esterbond against hydrolysis, such as a PEG-aromatic carboxyl ester,including a benzoic acid ester or a substituted benzoic acid ester.

In some embodiments, a more stable or slowly degrading bond such as aurethane bond or amide bond may be selected to provide the requisitemechanical strength and in vivo persistence to prevent the neuroma fromforming.

In other embodiments, hydrolysis modifiers (M) can be designed in thebackbone of the hydrogels to increase the hydrolytic degradation ofurethane in the hydrogel. This can be accomplished with the addition ofelectron withdrawing groups which accelerate the reactions.

In one embodiment, the hydrolysis rate of carbamate bond can bemodulated by the adjacent groups, thus modulating the persistence ofhydrogel in vivo. R1 and R3 can be any aliphatic hydrocarbon group(—CH₂—, —CHR—, —CRR′—), substituted aliphatic hydrocarbon group,aromatic groups and substituted aromatic group in any arranged forms.The aromatic group includes but not limit to phenyl, biphenyl,polycyclic aryls and heterocyclic aryls. The substitution moiety foraliphatic and aromatic group include but not limit to halogen, alkyl,aryl, substituted alkyl, substituted aryl, substituted heteroaryl,alkenylalkyl, alkoxy, hydroxy, amine, phenol ester, amide, carboalkoxy,carboxamide, aldehyde, carboxyl, nitro and cyanide. R2 can be H and anygroup in R1 and R3. In addition, R1 can include isocyanate, aromaticisocyanate, diisocyanate (e.g. LDI). In one embodiment, R3 can beAnilide and in another embodiment R1 can be phenyl.

In another embodiment, the hydrolysis rate of carbamate bond can bemodulated by the modulator at the beta position. The modulator can beCF₃PhSO₂—, ClPhSO₂—, PhSO₂—, MenPhSO₂—, MeOPhSO₂—, MeSO₂—,O(CH₂CH₂)NSO₂—, CN—, (Et)₂NSO₂—. In yet other embodiments, thesemodifiers can be adapted for use in PEG hydrogels containing amide,carbonate and urea linkages. Additional modifiers that affect thehydrolysis rate of the carbamate linkage are described in U.S. Pat. No.7,060,259, incorporated for reference herein. Additional cleavablecrosslinks are described in Henise et al (2019) In vitro—in vivocorrelation for the degradation of Tetra-PEG-hydrogel-microspheres withtunable b-eliminative crosslink cleavage rates. International Journal ofPolymer Science, incorporated in entirety. These modifier groups, M, canbe on the backbone itself or a nearby side chain such as with abeta-eliminative linker as described by D. V. S anti et al (2012)Predictable and tunable half-life extension of therapeutics agents bycontrolled chemical release from macromolecular conjugates. PNAS, 109(6)6211-6216 and US20170312368A1 incorporated herein for reference. In someembodiments, a soft chain extender is added, such as an aminoacid-peptide based chain extender with ester linkages. For example poly(phosphoester urethanes) with chain extenders containing phosphoesterlinkages. For example poly (DL, lactide) is a chain extender orpoly(caprolactone) to extend the PEG chain and add a soft segment.Preferably the molecular weight of the chain extender may be 0.5 kDa to5 kDa, preferably 1 to 2 kDa, more preferably 2 kDa. The soft segmentscan provide additional properties to enhance the physical properties ofthe hydrogel including the thermosensitivity, crystallinity, potentialresulting in physical in addition to chemical crosslinking. Thesehydrogels may be comprised of, for example, PEGs with molecular weightbetween 1,000 Da and 50 kDa including multi-arm PEG-succinimidylcarbonate (4-arm or 8-arm) with molecular weights of 5 to 40 kDa and armlengths between 1 and 3 kDa, and PEG-amine (4-arm or 8-arm) withmolecular weights between 5 to 40 kDa, preferably 10 or 20 kDa. In oneembodiment, PEG-SC (4-arm 10K) is crosslinked with PEG-amine (8-arm20k). In another embodiment, PEG-SC (8-arm 15K) is crosslinked withtrilysine amine. In another embodiment, PEG-SC (4-arm 20K) iscrosslinked with trilysine amine. Examples of other in situ formingPEG-SC formulations are described in U.S. Pat. No. 6,413,507,incorporated herein for reference. In another embodiment, a 4-arm PEGsuccinimidyl glutaramide 4-arm 10K (PEG-SGA) may used in combinationwith 8-arm PEG-amine 20K at 8% solid content.

Alternatively, the functionalized PEG urethanes and esters may becovalently crosslinked with another reactive polymer or small molecule(e.g. trilysine) containing amines or protected amines, maleimides,thiols or protected thiols, acrylates, acrylamides, carboxylic acids orprotected carboxylic acids, azides, alkynes including cycloalkynes, 1-3dienes and furans, alpha-hydroxycarbonyls, and N-hydroxysuccinimidyl,N-hydroxysulfosuccinimidyl, or nitrophenyl esters or carbonates.

In yet other embodiments, blends of faster degrading PEG-esters andslower degrading PEG-SGA or PEG-SC with ratios of 10:1 or 5:1 may permitslowing of the in vivo degradation profile. Similarly, blends ofmulti-arm PEG-SC and PEG-amine that crosslink to form carbamate bondsand PEG-carbonate ester bonds (delayed reaction of PEG-SC and withhydroxyl functional groups) to form blended 60:40 hydrogels (Kelmanskyet al (2017) In Situ Dual Cross-Linking of Neat Biogel With ControlledMechancal and Delivery Properties. Molecular Pharmaceutics, 14(10)3609-3616.

In yet other embodiments, multi-arm PEGs can be combined with blocks ofother hydrolytically degradable polymers that can be used to tailor thedegradation time of the PEG hydrogels. For example, soft segments withdiblock with polyester or triblocks can be synthesized with lowmolecular weight polyester regions to permit the hydrogel to be formedin an aqueous environment (polycaprolactone, polylactic acid,polyglycolic acid, polyurethane, polyhydroxyalkanoates (PHA),poly(ethylene adipate) (PEA), alipathic diisocyanates such as isophoronediisocyanate (IPDI) or L-lysine ethyl ester diisocyanate (LDI)). Theseblocks can be comprised of lactide, glycolide, or caprolactone regionscan, depending on the degree of crystallinity (D,L or L,L) be used toprovide additional mechanical strength to the hydrogels permit tuning ofthe degradation profile. For example, a block of caprolactone can beadded to a multi-arm PEG which each arm comprising a PEG-PCL-NHS ester.In this embodiment, the PCL domain may extend the degradation of apreviously poor in vivo persistent multi-arm PEG with hydrolytic esterlinkages. In the preferred embodiment, a PCL block of between 1 and 5kDa, preferably 1 to 2 kDa is added on the PEG arm. For example, a 4-arm28K PEG-PCL-NHS ester may react with an 4-arm 10K PEG-amine to form acrosslinked hydrogel in situ, where the PEG is a 2K block. The additionof the block renders the hydrogel in situ forming both through chemicaland physical crosslinking. Amino acids can also be incorporated as chainextenders in the PEG-SC to improve the degradation of the PEG-urethane.In some embodiments low molecular weight trifunctional polyester polyolsare selected for incorporation. Please refer to FIG. 1—common monomersused for synthesis of biostable and biodegradable polyurethanes,incorporated herein for reference (Chapter: Degradation of Polyurethanesfor Cardiovascular Applications, Book: Advances in Biomaterials Scienceand Biomedical Applications).

In some embodiments heterobifunctional crosslinkers are used to enablepolyesters to be conjugated to some arms and NHS esters or otherfunctional group with other arms.

In yet other embodiments, excipients may be incorporated into thehydrogels to modify the mechanical strength, density, surface tension,flowability, and in vivo persistence of the hydrogels. These modifiersare encapsulated in the hydrogel when the hydrogel is formed. Modifiersmay include amphiphilic excipients such as vitamin E TPGS, low molecularweight polyesters such a caprolactone or solvents such as ethanol. Inone embodiment, ethanol is incorporated in the diluent or acceleratorsolutions to yield a 5% to 70% v/v ethanol loaded hydrogel. The ethanolimproves the elasticity of the hydrogel and reduces the density of thehydrogel precursor solution relative to the nerve density. Further more,low concentrations of ethanol may be incorporated in the hydrogel toimprove the pot or functional life of the PEG/diluent solution after PEGpowder suspension. In another embodiment, Pluronic may be incorporatedin diluent or accelerator solution to yield a 5 to 15% w/v to yield aPEG-SG hydrogel with improved elasticity and in vivo persistence. In yetanother embodiment, low molecular caprolactone is incorporated intodiluent solutions to yield a 1 to 5% w/v PEG/caprolactone blendedhydrogel. In another embodiment, vitamin TPGS can be incorporated intothe diluent solution to yield a 5 to 20% w/v of PEG/vitamin E TPGSblend.

Swelling Another critical element of these hydrogels is the swelling ofthe hydrogels for applications around nerves. The hydrogels, whendelivered circumferentially around an object such as a nerve, undergopositive swelling in an outward radial direction. Initially, thehydrogels undergo equilibrium-mediated swelling as they equilibrate withthe fluids in the surrounding environment, and, later, when a criticalnumber of hydrolytic bonds have broken, the hydrogels swell as a resultof loss of mechanical strength. This latter phase ofdegradation-mediated swelling results in the progressive loss ofmechanical strength and hydrogel softening that the hydrogel collapsesand is ultimately cleared from the site. In vivo experiments in whichtransected rat sciatic nerves were surrounded within hydrogels thatswelled 5%, 10%, 20%, 30% and 60%, demonstrated that hydrogels thatswell more than 30% were significantly more likely to fall off of thenerve as a result of the creation of a gap between the nerve and thehydrogel. Of note, PEG hydrogels that swell at or less than 0%, shrinkwhen equilibrating in vitro or in vivo and the resultant compression mayresult in persistent hydrogel-mediated local nerve pain. For example,the DuraSeal hydrogel swells significantly and have a tendency to fallof the proximal nerve stump when delivered in situ.

Equilibrium swelling. For applications in which hydrogels are deliveredto nerves to prevent nerve regeneration, maintaining close adherence andapposition between the nerve and the conformable hydrogel is desirable.As a result, minimizing the equilibrium swelling post-hydrogel deliveryis desirable. The equilibrium swelling occurs during in the minutes todays as the hydrogel equilibrates with the fluids in the in situenvironment. In one embodiment, the hydrogel swells greater than 0% butless than 40%, preferably greater than 5% and less than 30%, morepreferably greater than 5% and less than 25%.

Furthermore, in some embodiments, it is desirable to avoid hydrogelsthat shrink as these hydrogels may compress the nerve and result inaberrant nerve firing and therefore it is preferable to use hydrogelsthat swell greater than 0%. In addition, the nerve may swell afterinjury, and so some swelling is desirable to permit some space for thenerve to swell.

Equilibrium swelling may be preferably assessed in vitro at bodytemperature conditions (37° C. in PBS). Hydrogel samples were preparedin cylindrical silicone tubing (6 mm) and cut to dimensions of 6 mmdiameter by 12 mm length. Samples were weighed and merged into PBS at37° C. After swelling in PBS for 12 to 24 hours at 37° C., samples weretaken out and weighted again. The swelling is calculated by thepercentage of mass increase.

Degradation swelling. A secondary characteristic in biodegradable orbioerodible hydrogels, after the initial equilibrium swelling, is anappreciation for a second ongoing phase of swelling that occurs as aresult of the degradation of the hydrogel. The swelling may occurthrough the hydrolytic, enzymatic, or oxidatively-sensitive bonds in thehydrogel. This is an equally important characteristic because thehydrogel needs to remain on the nerve for a period of one month or more,more preferably two months or more, more preferably three months. In ananimal model, the period of time is shorter and in the clinical settingthis period is longer. In some instances, if the degradation rate is toorapid, the hydrogel may fracture and fall off the nerve or be clearedbefore the hydrogel can serve the function to prevent nerve outgrowthand/or neuroma prevention. In other instances, if the hydrogel appearsintact on the nerves, there may be a substantial loss of the mechanicalintegrity within the hydrogel as a result of degradation that the nervemay extend out into the softened or fractured hydrogel and form aneuroma formation. As a result, it is preferable that a biodegradablesystem have no more than 50% of the hydrolytically labile linkagescleaved at 3 months, more preferably no more than 30% of the linkages,and even more preferably no more than 20% of the linkages. After theperiod of time in which the hydrogel provides a mechanical barrier tonerve regeneration, the crosslinking density can drop and thedegradation can continue until the hydrogel is entirely cleared. Theloss of bonds can be evaluated in part through the reduction in themechanical integrity of the hydrogel. The loss of bonds can be evaluatedin part through the reduction in the mechanical integrity of thehydrogel. Thus, it is desirable that the hydrogel maintain a compressionmodulus of 40 kPa at 3 months post delivery, this hydrogel issufficiently stiff that nerves will not grow through it.

Pressure. In addition to ensuring that the swelling is not so great thatthe hydrogel falls off of the nerve, confirmation that the swelling (orlow swelling, shrinkage) will not result in nerve compression is alsodesirable. In one experiment, a pressure transducer catheter was placednext to a nerve and an in situ forming hydrogel was delivered to formaround the nerve/pressure transducer (Millar Mikro-Tip pressurecatheter, 3.5 F, single straight, AD Instruments). The hydrogel was thenplaced at 37° C. in PBS and measurements of the pressure as a functionof time were taken. Hydrogels with approximately 0% or negative swellingresulted in high and sustained increases in the pressure (>80 mmHg)exerted on the embedded nerve whereas hydrogels that swelled 10% or moredid not result in any significant increases in pressure (˜20 mmHg). Inthe preferred embodiment, the pressure reading after equilibriumswelling is around 5 mmHg In preclinical and clinical models, pressureat the site of nerve damage may be between 5 and 15 mmHg (Khaing et al2015—Injectable Hydrogels for Spinal Cord Repair). For example, althougha variety of materials have been evaluated for modulating nerveregeneration in a spinal cord model, the majority do not have therequisite linear compressive modulus (G) to prevent neuroma formation(Table 1, Khaing et al, 2015).

Stiffness. Stiffness of the hydrogel can measured/inferred either byrheology (G′=storage modulus, G*=shear modulus, or the linearcompressive modulus (G). Preferably the stiffness of the hydrogels, asmeasured through the linear compressive modulus (G) is greater than 10kPa, preferably greater than 30 kPa, more preferably greater than 50kPa. The stiffness prevents nerve outgrowth into the surroundinghydrogel.

Compression and rebound. In addition to injectable gels that haveminimal swelling, gels that are compressible are desirable. In thismanner, even if the hydrogel implant is pressed, it will not fracture.Compression and rebound testing is performed on cylindrical samples (6mm diameter, 10 mm long) that have been incubated for at least one hourat 37° C. until equilibrated. The samples are loaded into the Instronand a displacement perpendicular to the longitudinal axis of thecylinder will be applied at a crosshead speed of 1 mm/min to a finaldisplacement of 60% of the diameter of the conduit. Verification thatthe hydrogels can withstand compressive forces of greater than 0.25N andthat no changes in the shape and diameter have occurred after removal ofthe compressive forces.

Flexibility. Another critical parameter of these in situ formingpolymers is the ability of the hydrogels to bend and flex atphysiologically relevant angles in the body. To evaluate the flexibilityof the hydrogels, the hydrogels were formed inside 0.1 to 0.25″ innerdiameter silicone tubing to form 12 to 24″ long cylindrical hydrogelcables. Preferably, the hydrogels have sufficient flexibility to bendgreater than 90° and more preferably cylindrical strands of hydrogel canbe readily be tied into a knot. Since the flexibility and elasticity isdetermined, in part, by the distance between the core of one multi-armPEG to the core of the adjacent multi-arm PEG, PEG hydrogels withcore-to-core distances of 3 kDa, more preferably 5 kDa or more. Flexiblerobust hydrogels that will not fracture in the highly mobile andcompressive environment of the body. As a result, more flexiblehydrogels are desired such as combinations of the 4-arm 10K or 20 K PEGswith 4-arm or 8-arm 20K PEG-amines may be desirable.

Viscosity. Low and medium viscosity precursor solutions may be selectedto encapsulate the hydrogel with generally better adhesion in the lowviscosity solution and improved handling of the nerve in the mediumviscosity precursor solutions. In one embodiment of the invention, theflowable media is a low viscosity hydrogel precursor solution, having aviscosity of no more than about 100 cP and in some embodiments no morethan about 20 cP or no more than about 5 cP. In yet another embodiment,the flowable media is a medium viscosity hydrogel precursor solution,having a viscosity preferably 300 to 10 kcP, more preferably 300 to 900cP. In one embodiment, a viscosity enhancer/modifier or thickening agentcan be added to the gel precursor to modify the fluidic properties andhelp to positioning the nerve in the cap before gelling. The viscositymodifier can be natural hydrocolloids, semisynthetic hydrocolloids,synthetic hydrocolloids and clays. Natural hydrocolloids include but notlimited to Acacia, Tragacanth, Alginic acid, Alginate, Karaya, Guar gum,Locust bean gum, Carrageenan, Gelatin, Collagen, Hyaluronic acid,Dextran, Starch, Xanthan gum, Galactomannans, Konjac Mannan, Gumtragacanth, Chitosan, Gellan gum, Methoxyl pectin, Agar, Gum arabic,Dammar gum. Semisynthetic hydrocolloids include but not limited toMethylcellulose, Carboxymethylcellulose, Ethyl cellulose, Hydroxy ethylcellulose, Hydroxy propyl methyl cellulose (HPMC, 0.3%), Modifiedstarches, Propylene glycol alginate. Synthetic hydrocolloids include butnot limited to Polyethylene glycol, Polyacrylic acid, polyvinyl alcohol,polyvinyl pyrrolidone, polyglycerol, Polyglycerol polyricinoleate. Claysinclude but not limited to Magnesium aluminum silicate (Veegum),Bentonite, Attapulgite.

In another embodiment, viscosity can be modified by pre-crosslinkingPEG-amine and PEG-urethane. PEG-amine and PEG-urethane canpre-crosslinked at a ratio from 10000:1 to 1:10000 at total PEGconcentration of 0.01% to 100%. The pre-crosslinking can be furthercross-linked by itself, or with PEG-amine, or with PEG-urethane, or withPEG-amine and PEG-urehtane to form a higher viscosity precursorsolution.

Density of precursor solution. Nerve tissue, as a result of the myelinand adipose tissue, is hydrophobic and thus has a tendency to float insolutions with density approximating that of water (˜1 g/cm³). Byadjusting the density of the flowable media, the nerve position can beadjusted to reduce the propensity of smaller diameter nerves to float upin the precursor solution without sacrificing adhesion strength thatcomes with increasing precursor viscosity. In some embodiments, thedensity of the precursor solution or media solution is decreased so thatthe nerve is relatively more dense than the solution to <1 g/cm³,preferably <0.9 g/cm3. In still other embodiments, the density of theprecursor solution is adjusted to be approximately equivalent to that ofthe nerve. Polar and less dense solvents can be added including ethanol(10 to 70%), toluene (10%), ethyl acetate or chlorobenzene to reduce thedensity of the precursor solution. In another embodiment, vitamin E TPGS(1-2 kDa, 10-20%) can be added to reduce the propensity of the nerve tofloat up. Some of these solvents also reduce the surface tension of theprecursor solution, causing the nerve to sink.

Open Surgical Neuroma Procedure. After openly exposing the surgical siteand isolating the target nerve, fresh transection of the nerve isdesirable to clean up the nerve end. In some embodiments, the clinicianmay elect to transect the nerve at a 90 degree or, alternatively, a 45degree angle. In some embodiments, the clinician may elect to use othermethods such as electrocautery of the nerve end, ligation of the nervestump, apply low molecular weight end-capped PEG (e.g. 1-5 kDa, 50 w/v %hypotonic solution) or other approaches that they have developed to sealor ablate the end of the nerve.

Axoplasm. As axoplasm, a viscous and sticky material that oozes from theend of the cut nerve after transection, may reduce close appositionbetween the nerve end and the hydrogel, it may be desirable to remove itfrom the nerve tip. This can be accomplished through the contact betweenthe nerve and an absorbent material, such as a swab. An absorbable swabmay be provided in order to absorb any of the axoplasm after nervetransection. The tip of the swab is preferably less than 5 mm, morepreferably less than 2 mm in order that it may fit comfortably withinthe form and hold the nerve while the hydrogel is delivered. The swabmay be provided as part of the kit. Alternatively, this can beaccomplished by contacting the nerve tip with the surrounding tissue,which results in the rapid formation of an adhesion between the nerveand the tissue that must then be secondarily severed.

Coverage of the proximal nerve stump. The hydrogel itself preferablyextends at least 0.5 mm, preferably 1 mm to 20 mm, preferably 2 mm to 10mm beyond the end of the proximal nerve stump.

Length coverage. It is preferable that a minimum of 10 mm of nerve beembedded/encapsulated in the hydrogel although, in some instances, 5 mmmay be sufficient. A greater length of nerve embedded in the hydrogelaccomplishes several things a) increased surface area of appositionbetween the nerve and the hydrogel and b) decreased likelihood ofproximal sprouting from nodes of Ranvier proximal to the transection asthese proximal nodes are embedded in hydrogel. Again, the greater thelength of nerve encapsulated, the higher the likelihood that even theregions that were damaged through handling with forceps, previoustrauma, etc. are embedded in the hydrogel, thereby preventing sproutingof nerve fibers.

Once an approximately 10 mm section of nerve is isolated, whether thenerve is adherent to the swab, the side of a forcep or rod, or is gentlyheld by a pair of forceps, the nerve is elevated slightly to allow aform to be placed underneath the nerve. In one embodiment, the nerve isthen gently lowered into a channel or entry zone to align the nerve inthe center of the form. See, e.g. FIGS. 1A and 1B. The form, once thedesirable position is reached, is left in place.

While holding the nerve tip in one hand in the center of the form, theclinician then delivers the in situ forming hydrogel using the otherhand to fill the form and retain the nerve in the center of the form.The top of the silicone form serves as a guide for when to stop fillingthe form. As the hydrogel fills past the top of the nerve, theswab/forcep is removed so that the nerve is retained within the hydrogeland not the tool. In this manner, there is no direct path for a nerve toregenerate through the surrounding tissue and the nerve is completelysurrounded by the hydrogel.

Gel thickness. Preventing nerve regeneration requires providing aconformable barrier at the proximal end of a transected nerve. Thehydrogel also preferably surrounds the nerve with a thickness of 100 μmto 5 mm radially. In one embodiment, the hydrogel precursor solution isdripped over the nerve to form a thin protective coating approximately100 microns to 2 mm in thickness circumferentially around the proximalnerve stump and remain in the place to block neurite outgrowth. A thincoating is sufficient to provide a barrier to nerve regeneration andthus in some embodiments, the nerve is dip-coated in the flowable mediaand the hydrogel subsequently forms in a thin layer around the nerve. Inthis embodiment, the hydrogel gelation time is adjusted to 10 to 20seconds to permit the removal of the coated nerve prior to theconversion to a nonflowable form with gel formation. Thin coatingsproviding adhesion to and coverage of the circumference and tip of thenerve stump on the order of 50 microns to 500 microns to cover the endof the nerve are desirable.

In yet another embodiment, it is desirable to form the hydrogel aroundthe nerve in an implant or bolus, providing a robust adhesive layer ofhydrogel around the nerve, approximately 0.5 to 5 mm, more preferably1-2 mm in thickness around the circumference of the nerve and 1 to 5 mmbeyond the tip of the nerve stump tip.

Pore size. In order to prevent nerve regeneration into the biomaterial,the pore size of the hydrogel needs to be sufficiently small to preventnerves and other supporting cells from infiltrating the biomaterial.Nerve axon diameters are between approximately 0.5 and 30 μm.Preferably, the growth inhibitory hydrogel is microporous or mesoporous,with pores less than 1 μm, preferably less than 0.5 microns, morepreferably less than 500 nm in diameter.

Charge Neutrally or negatively charged biomaterials are preferred asgrowth inhibitory gels as neurites prefer to grow into positivelycharged biomaterials. Similarly, hydrophilic materials or amphiphilicare preferable to hydrophobic materials.

Nondegradable hydrogels. If a nondegradable hydrogel system is used, thesame equilibrium swelling characteristics apply but, since the hydrogelis nondegradable or biostable, degradation swelling is not relevant. Forexample, the nondegradable in situ forming thermoresponsive copolymer,Pluronic (PEO-PPO-PEO), polyvinyl alcohol, or PEO may be utilized toform hydrogels.

Clarity. In the preferred embodiment, the hydrogel is clear andtransparent to confirm the location of the nerve after hydrogelformation. A visualization agent may be incorporated in the hydrogel toaid in contrast relative to the background tissues. The color additiveor color additive blend may be included in a the polymer powdersolution. In the case of the use of multiple hydrogels (describedbelow), the use of one or more different visualization agents isdesirable to provide visual confirmation, for example, that the growthpermissive hydrogel was correctly delivered between the nerves and thegrowth inhibitory hydrogel was delivered around the growth permissivehydrogel. Elasticity. In some embodiments, the elasticity of hydrogelcan be modulated by incorporating hydrophobic domain into the hydrogel.The hydrophobic domain can be incorporating by crosslinking or mixing ofmolecules, particles, fibers and micelles. The molecules incorporatedcan be amphiphilic molecules including pluronic, polysorbate andtocopherol polyethylene glycol succinate. The particles, fibers andmicelles can be made from amphiphilic molecules described in the abovesection. In addition, many low molecular weight hydrophobic drugs thatare incorporated into the hydrogel (described below) improve theelasticity of the hydrogel.

Kit. The in situ forming hydrogel is delivered through a dual applicatorsystem comprising a dual channel applicator, a dual adapter, one or moremixers, and one or more blunt needles. Also included in the kit is apowder vial with associated vial adapter, diluent solution, andaccelerator solution for use in the dual applicator system. The kit mayinclude one or more forms into which the hydrogel is delivered. The kitmay also include on or more mixer-blunt syringes. The Mixer syringes maybe conventional single lumen mixers with a static mixing element or themixers may be mixers in which there is recirculation and turbulent flowof the contents to improve the mixing of the precursor solution.

Chemical and physical. Preferably, the hydrogel networks arepredominantly hydrophilic with high water content and are formed throughthe physical or chemical crosslinking or synthetic or natural polymers,copolymers, block copolymers or oligomers. Examples of synthetichydrogels that are not growth permissive include agarose, PEG, oralginate, PVA hydrogels with 2% w/v solid content or higher, preferably6% w/v solid content, more preferably 8% w/v solid content or higher.PEGs. Multi-arm PEGs are described above but may be selected accordingto the desired properties from PEG-amine, PEGarboxyl, PEG-SCM, PEG-SGA,PEG-Nitrophenyl carbonate (carbonate linker), PEG-Maleimide,PEG-Acrylate, PEG-Thiol, PEG-Vinysulfone, PEG-Succinimidyl Succinate(SS), PEG-Succinimidyl Glutarate (SG), PEG-Isocianate, PEG-Norbornene orPEG-Azide. Alginate. Viscous injectable alginate sol (1 to 5%) may bedelivered around the nerve. Similarly, agarose gel at concentrations of1% wt/vol or more prevent nerve extension.

Hypotonic solutions. In one embodiment, a hypotonic solution (Ca2+ free,slightly hypotonic, saline solution containing 1-2 mM EGTA) is deliveredto the cut nerve to assist in the sealing of crushed or transectedaxonal ends prior to repair with the in situ forming biomaterial.

PEG Fusion Combined with an In Situ Nerve Cap or Wrap. As described inthe many publications outlining methods for PEG fusion of nerves (3.35-5kDa, 30-50% w/v solution), a PEG solution can be delivered to the nervesto first fuse the nerves, alone or in combination with methylene blue.After sealing the membranes, the growth permissive hydrogel is deliveredin between and around the compressed or severed nerves.

Cross-linked Particles. In some embodiments, the hydrogel can be madewith cross-linkable particles, fibers, or micelles. These particles,fibers or micelles are functionalized with reactive groups, includingbut not limited to active ester, amine, carboxyl, aldehyde, isocyanate,isothiocyanate, thiol, azide and alkyne, which can be cross-linked withsmall molecules, polymers, particles fibers or micelles with reactivegroups to form bonds including amide, carbamate, carbonate, urea,thiourea, thioester, disulfide, hydrazone, oxime, imine, amidine andtriazole. In some embodiments, the micelles, fibers and particles can beformed from amphiphilic macromolecules with hydrophilic and hydrophobicsegments. The hydrophilic segments can be natural or synthetic polymers,including polyethylene glycol, polyacid, polyvinyl alcohol, polyaminoacid, polyvinyl pyrrolidone, polyglycerol, polyoxazolines, andpolysaccharides. Hydrophobic segments can be fatty acids, lipids, PLA,PGA, PLGA, PCL and the polymer ester copolymer at different ratio. Inanother embodiment, functionalized microparticles form physicalcrosslinks with one another after a pH change, and then, when placed insitu, the functionalized particles crosslink to form an interconnectednetwork of microparticles.

Sealants. Some of the in situ forming gels developed for adhesionprevention and sealants may also be adapted for this application topreventing neuromas and aberrant nerve outgrowth into scar tissue, suchas low molecular weight polyanhydrides of acids like sebacic acid,including poly(glycerol-co-sebacate) (PGSA) based sealants (U.S. Pat.No. 9,724,447, US20190071537, Pellenc et al (2019) Preclinical andclinical evaluation of a novel synthetic bioresorbable, on demand lightactivated sealant in vascular reconstruction, incorporated herein andadapted for use around nerves, for reference). Another sealant that maybe adapted for delivery around peripheral nerves is the Adherus DuralSealant, which comprises a PEG-polyethylenimine (PEI) copolymer thatforms in situ, as it exhibits low swelling and degrades in about 90 days(U.S. Pat. No. 9,878,066, incorporated herein). Other sealants includeBioGlue Surgical Adhesive (Cryolife), composed of bovine serum albuminand glutaraldehyde, Omnex (Ethicon), ArterX (Baxter), Coseal (Baxter)and TissuGlu, composed of lysine based urethane (Cohera Medical).

Photoresponsive. In some embodiments, photoresponsive, photopolymerizingor photocrosslinked biomaterials are envisioned that could be deliveredin a liquid (low to medium viscosity) state into the form (cap or wrapform) around the nerve, and then, when the proper positioning of thenerve is obtained within the form, the photopolymerization is initiatedwith either ultraviolet or visible light. In one embodiment, the lightsource can be attached directly or via fiber optic cable that interfacesdirectly with an opening in the cap or wrap form. The cap form diffractsthe light such that it ensures that the entire form is sufficientlyilluminated to achieve consistent homogenous crosslinking across thegel. In the preferred embodiment, the light source housing is coupleddirectly with the form at the distal end of the cap facing the nervestump face to ensure that the light directly penetrates. Alternatively,the form may be designed with embedded light-emmitting elements thatpermit the light to be delivered circumferentially around the nerve.Hydrogels include PNIPAAM hydrogels modified with a chromophore such astrisodium salt of chlorophyllin.

Other forms. In addition to a cross-linked network, hydrogels may becomprised of dendrimers, self-assembling hydrogels, or low molecularweight synthetic polymer liquids.

In one embodiment, lower molecular weight hydrogels (2 kDa, liquid) canbe formed in situ without water as a solvent as described in Kelmanskyet al (2017) In Situ Dual Cross-Linking of Neat Biogel with ControlledMechanical and Delivery Properties), Molecular Pharmaceutics, 14(10)3609-3616, incorporated herein. In yet other embodiments, hydrogels canbe photocrosslined to form hydrogels, as extensively described in theliterature. Crosslinking agents include eosin,) In yet otherembodiments, electroconductive hydrogels are usied includingpoly(3,4-ethylenedioxythiophene) (PEDOT), poly(pyrrole), polyaniline,polyacetylene, polythiophene, ester derivative,3,4-propylenedioxythiophene (ProDOT), natural or synthetic melanin,derivatives, and combinations thereof.

Addition of sulfated proteoglycans. In some embodiments, it may bedesirable to deliver inhibitory environmental cues to the nerves inaddition to the mechanical barrier provided by the hydrogel. This can beaccomplished by the addition of inhibitory molecules and/orextracellular matrix to the hydrogel either through physical blending orchemical crosslinking. Sulfated proteoglycans, such as side chains witha negative charge such as glycosaminoglycans are of interest. Ofparticular interest is dermatan sulfate (DS).

Blends. In some embodiments, it may be desirable to create blends of twoPEGs to improve the degradability of the system. In one embodiment, thePEG-SC is combined with PEG-SG prior to crosslinking with trilysineamine to create a hydrogel that has sufficient mechanical support toprevent nerve outgrowth but then degrades more rapidly than PEG-SC. Thepersistent time of gel in vivo is fine-tuned by the ratio of PEG-SG andPEG-SC. With the increase of PEG-SC content, the persistent time of gelin vivo increases. In another embodiment, the PEG-carbamates are blendedwith the PEG-carbonates. Other hydrogels include PEG hydrogels comprisedof carbamate derivatives (U.S. Pat. No. 7,060,259).

Incorporation of agents. In some embodiments, agents can be dissolved orsuspended in the diluent or accelerator solution and surfactants orethanol can be added to stabilize the suspension. The drug can be alsoencapsulated in microparticles, nanoparticles or micelles and thensuspended in diluent or accelerator. In some embodiments, the hydrogelcan be made with cross-linkable particles and micelles. These particlesor micelles have reactive groups such as the active ester, amine,carboxyl, thiol and those described in U.S. Pat. No. 7,347,850 B2 andcan be crosslinked with small molecules, polymers, particles or micelleswith reactive groups which reacts with the former particles or micellesand forming bonds including amide, carbamate, carbonate, urea, thiourea,thioester, disulfide, hydrazone, oxime, imine, amidine and triazole. Inother embodiments, gel can be form by the swelling of particles. Thelarge volume of swelling can increase the particle contact and lock theminto their location to form gel.

Solid content. The solid content can be adjusted to fine tune theswelling and tensile properties of the hydrogel. For example, the solidcontent can be adjusted above the critical gelation concentration, suchas between 6 to 15% loading, more preferably 7-9% loading, morepreferably 8-8.5% solid content.

Crosslinking. Hydrogels may be formed in situ throughelectrophilic-nucleophilic, free radical, or photo-polymerization.

In vivo Persistence. In some embodiments a longer in vivo persistencemay be preferred, in which the hydrogel remains in situ for between 3months and 3 years, more preferably 6 months and 18 months, morepreferably 6 months and 12 months.

Adhesion. Adhesive strength is an important criterion for maintainingthe hydrogel in close apposition to the nerve. Adhesion may occurthrough crosslinking reactions between the hydrogel and the primary andsecondary amines on the tissue surface e.g. the epineurium or the aminegroups found on the surface of nerves, glia, and associated cells. Theadhesion strength should be greater than 10 kPa, preferably greater than50 kPa, more preferably greater than 100 kPa. Adhesive strength onnerves can be estimated by embedding the sciatic nerve in the hydrogels.The ends of the nerves are embedded in superglue between sandpaper andplaced in titanium clamps in a Bose ElectroForce 3200-ES. Nerves arepulsed at a rate of 0.08 mm/s until failure. Care was taken to ensurethat the nerves were used shortly after harvest and that the hydrogeland nerve were equilibrated in PBS at 37° C. prior to testing.

Other hydrogels. In situ forming polyanhydrides are also of interest fordeveloping applications directed towards nerves. In one embodiment,polyanyhydride polymers can be acrylated so that they can form in situthrough free radical polymerization. Alternatively, they can formthrough photocrosslinking. At lower concentrations, the polymers arewater soluble e.g. 10%. The prevention of nerve regeneration isconferred in part through their hydrophobicity. Incorporated forreference are US20180177913A 1, U.S. 62/181,270, and US201562181270P.

Applicator. Dual channel applicators used to deliver the in situ forminghydrogels are commercially available (Nordson Medical FibrijetBiomateral Applicators, Medmix Double Syringe Biomaterial DeliverySystem, K-System). However, these mixers, delivering between 2.75 and upto 10 ml of hydrogel, include a single lumen with a static mixingelement and are designed for the adequate mixing and delivery of largevolumes of hydrogel solution and are not ideal for delivering smallvolumes (<1 ml) of hydrogel solution to a site. As a result, there is aneed for a mixer that provides mixing of small volumes of two componentsystems as inevitably, one of the two solutions is typically advancedslightly ahead of the other solution, leading to a small volume ofpartially or inadequately mixed gel existing the needle first. In oneembodiment, a custom mixer is designed to fit onto the Nordson Medicalor K-System applicator, through either a luer or a snap fit, as the dualchamber applicator system necessitates, to recirculate some of theinitial solutions that enter the mixer in order to ensure better mixingof the hydrogel, including the initial components. FIG. 18 illustratesthe design (transparent) of the center portion of the mixer containingan entrance port with at least one static mixer, a larger containerthrough which the contents are delivered and recirculated and a secondport which captures the mixed recirculated fluid and delivers it to theexit port of the mixer. The second port may or may not containadditional static mixers.

Example 1

In some embodiments the 4-arm-PEG 10K-SC is crosslinked with an 8-armPEG 20K amine. The PEG-SC and PEG-amine were dissolved in an acidicdiluent at a ratio of 1:1. The suspension was mixed with acceleratorbuffer and delivered through a static mixer to form a hydrogel. Thisformulation gelled in 4 seconds.

Example 2

In other example, 8-arm 15K PEG-SC is crosslinked with trilysine. ThePEG-SC was suspended in buffered trilysine solution and then mixed withaccelerator buffer through a static mixer. This formulation gelled in 2seconds and the gel provided compression strength up to 200 kPa.

Example 3

In other example, 8-arm 20 K PEG-thioisocyanate is crosslinked withtrilysine at a ratio of 1:1. The formulation gelled in 3 seconds and hasa compression strength of 120 kPa and 5% swelling.

Cap Form. The method may comprise the step of positioning a form at atreatment site before the positioning the severed end step. The form isprovided as part of the kit containing the delivery system and iscomposed of an inert, biocompatible, flexible and nonadhesive materialto provide the desired shape to the in situ formed material. The form isdesigned to be filled with the flowable media such that it flows aroundthe proximal nerve stump end where it transforms to a non flowablecomposition, conforming to the nerve stump and preventing neuromaformation. In the preferred embodiment, the form creates a low profilenerve cap with a smooth transition between the nerve and the cap andapproximately cylindrical shape around the nerve.

Shape. A form is desirable, not only because it reduces off targetspread of the in situ forming material but because it provides alow-profile circumferential lubricious shape that cannot be achievedwith the application of the hydrogel alone. The profile and transitionsof the form design reduce the friction and interference with thesurrounding tissue, allowing the hydrogel to glide and rotate relativeto the surrounding tissue. The cap is designed to be able to cover atleast 5 mm, preferably a 10 mm length or more of nerve.

In accordance with another aspect of the present invention, there isprovided a form for creating a formed in situ nerve cap to inhibitneuroma formation. The form comprises a concave wall defining a cavity,the wall having a top opening for accessing the cavity. The top openinglies on a first plane and has an area that is less than the area of asecond plane conforming to inside dimensions of the cavity and spacedapart into the cavity and parallel to the first plane. A concave nerveguide is carried by the wall and provides a side access to the cavityfor receiving a nerve end. The wall is flexible so that it can beremoved from a crosslinked nerve cap formed within the cavity, and maycomprise silicone, preferably with a durometer of 30-50, most preferablya durometer of 40. The wall design of the form has a slight undercutsuch that the material, when filling the form to the top edge of theform, forms a convex surface due, in part, to surface tension of themedia, that completes the cylindrical shape of the hydrogel.

Silicone. In one embodiment, the form is comprised of a nonadherentnondegradable material. In the preferred embodiment, the material ismedical-grade silicone, sufficiently flexible to be peeled or popped offof the in situ formed material (e.g. durometer 40). After the transitionof the media to a substantially non-flowable state, the silicone form isremoved and discarded. In one embodiment, the silicone form is coloredto provide contrast against the surrounding tissue so that thenondegradable polymer is not accidentally left in situ. Darker colorsare preferable to enhance the light that enters the cap and illuminatethe nerve, such as dark blue, dark purple or dark green.

Biodegradable. The method may alternatively comprise the step of placinga biodegradable form before the positioning the severed end step. Thebiodegradable form may be comprised of a non-crosslinked lyophilized (ordried) synthetic biomaterial that remains in place for approximately 5to 10 minutes during the time that the in situ forming hydrogel isdelivered, and then is rapidly dissolved and cleared from the site inless than, for example, one or two days. In one embodiment, thebioerodible form is comprised of lyophilized multi-arm end-capped or noncrosslinked PEG, lyophilized linear PEG (3.35 kDa) or crosslinkedmulti-arm PEG (e.g. 8-arm 15 kDa). The method may alternatively comprisethe step of forming a biodegradable form in situ before the positioningthe severed end step. In alternate embodiments, the form is composed ofmaterials typically used for nerve conduits and wraps, such as polyvinylalcohol, chitosan, polylactic acid, polyglycolic acid, polycaprolactone.

In yet another embodiment, the ex vivo implantable form is comprised ofthe same material as the in situ formed material that is delivered intothe form. In this manner, the properties of the equilibrated form arecomparable and match well with the equilibrated in situ formed hydrogel.In these embodiments, the biodegradable form for remains in place afterdelivery of the hydrogel and is not removed but is cleared from theimplant site at approximately the same rate as the in situ formedmaterial. In yet another embodiment, the form is comprised oflyophilized non-crosslinked PEG into which the in situ formed hydrogelmedia is delivered. The non-crosslinked PEG is readily solubilized andcleared from the site, making the form a rapidly bioerodible form. Inyet another embodiment, the form is comprised of a crosslinked PEGmatrix that will be cleared rapidly from the site as a result of arapidly cleaved hydrolytic bond, such as can be obtained with the esterlinkage in a PEG succinimidyl succinate (PEG-SS).

Forms can be synthesized by injection molding, crosslinking orpolymerizing in a cavity, solvent casting, or 3D printing. A range ofsynthetic and natural materials can be selected for the implantableform, including collagen, PEG-PEI, alginate, chitosan, or agarose.

Forms may be rapidly dissolving forms, that, upon wetting, dissolve andare cleared within an hour so after the procedure, leaving the in situformed biomaterial in place. Alternatively, forms may be more slowlydegrading forms that swell to a similar or greater extent that the insitu formed material that is delivered inside them. The swellingprevents scenarios in which the hydrogel swells during equilibriumswelling and compresses the nerve if the form into which it is deliveredis shrinking or has minimal compliance.

In some embodiments, the nerve positioned is held in the desiredlocation or orientation with forceps in one hand and the media isdelivered with the other hand into the form. As the media fills theform, the nerve is released and subsequently the media changes to anon-flowable state. Alternatively, a supporting physician or nurse mayassist with the procedure. In another embodiment, the growth inhibitoryhydrogel is formed around the nerve in a two-step process. In the firststep, the hydrogel is delivered to the nerve tip to encapsulate thenerve end. In the second step, the hydrogel is applied to fill theentire form, including the nerve tip. In another embodiment, the growthinhibitory hydrogel is formed around the nerve in a first layer and thena second layer of hydrogel is subsequently applied, in a two-stepprocess.

Conformability. Unlike wraps, which still have a gap between the wrapand the nerve, the hydrogel conforms directly to the nerve itselfproviding a barrier to inflammatory and pro-scar forming cells to thesite while allowing nutrients through. Because the hydrogel adheres tothe nerve, there is no need to suture the nerve to the hydrogel. Theproximity of the hydrogel to the nerve also helps to prevent scar andadhesion formation around the nerve in the initial healing phase.

Centering. Nerves, by virtue of their low density and high fat contentand flexibility, particularly smaller nerves, have a tendency may flowupwards in a low viscosity solution. The following embodiments aredesigned to assure that the nerve does not float up after delivery ofthe media to the top of that surface.

Viscosity. As described above, the viscosity of the flowable solutioncan be increased to minimize the nerve floating up within the solution.

Flow. In another approach, the needle that delivers the flowable mediais directed in such a way that the flow of the media permits thecircumferential spread of the solution around the nerve prior to the gelforming. The cap form can also be designed to improve the flow dynamicsof the media and improve nerve alignment. In one implementation of theinvention the severing a target nerve step and the positioning a form ata treatment site step are accomplished by a single instrument. Inanother implementation of the invention, the nerve cap form is designedsuch that the delivery system and the form are integrated. In thepreferred embodiment, the delivery system is connected to the form via acatheter. The catheter entrance in the cap resides at the same entrancewhere the nerve is entering the form. The catheter permits the flow ofmaterial down the shaft and circumferentially around the nerve such thatthe media acts to self-center the nerve within the form.

Stabilizer. In another embodiment, a stabilization rod or piece isaligned directly under the nerve or against the nerve such that itprovides sufficient adhesive forces that the hydrogel can be deliveredaround the centered nerve into the form.

Peelable conduits In yet another embodiment, the nerve is positioned inthe center of the in situ biomaterial through the consecutive placementwithin two peelable conduits. In brief, the nerve is placed inside thefirst peelable conduit and the in situ forming material is delivered tosurround the top and distal end of the nerve. The peelable conduit maybe an open ended or close ended conduit. After the hydrogel forms, thesheath is pulled apart along weaker peel lines in the material andiscarded. The resultant nerve-hydrogel is then placed in a secondlarger peelable conduit. By rotating the nerve slightly, the hydrogelsurface can be placed on the bottom of the second sheath so that thenerve is centered in approximately the middle of the second sheath. Asecond application of the in situ forming hydrogel results in thecumulative formation of a circumferential hydrogel around the nervewhich protects and centers the nerve within the nerve cap. In oneembodiment, the sheath is composed of an extruded splittable PTFE tubewith a vertical tab to assist in tearing the piece apart in the surgicalsetting, similar to vascular introducers.

In another embodiment, the nerve is placed so that the proximal stumprests at a ninety degree angle downward in a cup-shaped form and thehydrogel delivered into the cup-shape to form around the nerve. Thecup-shaped form is subsequently removed and discarded and the proximalstump is adjusted back to its resting position in the tissue.

In yet another embodiment, the nerve can be delivered in an amphiphilicor hydrophobic solution to prevent the nerve from floating to thesurface of the medial. In yet another embodiment, the in situ formingmaterial may be more viscous, to prevent the nerve from migrating withinthe form.

Tilted. Alternatively, the form may have a tilt in the form, to bias thenerve filling from the distal to the proximal end. In this manner, thenerve can be positioned in such a way that the hydrogel formscircumferentially around the proximal nerve tip first and then, eitherthrough a second application or a continuation of the first application,fills the rest of the form.

Entrance centering. In one embodiment, the forms are designed in such away that the nerve enters at a lower level relative to the top of theform to permit the material to be delivered circumferentially around thenerve. In another embodiment, the entrance region of the form is slopedso that the nerve enters the form at a downward angle, biasing theproximal nerve tip location downward.

Ribs. Tabs or ribs are provided on the external face of nondegradabletemporary forms, such as silicone forms, to assist with the removal ofthe form after formation of the gel. These tabs are placed in such aplace to provide additional stability for the cap form on irregularsurfaces or to provide a surface to grip with forceps or other surgicalinstruments. In yet another embodiment, the form is designed to be selfcentering. In other words, the form may naturally seat itself so thatthe top surface of the form is level in preparation for delivery of thein situ forming hydrogel.

Holes. In some embodiments, a hole or guidance sheath is provided todirect the needle to deliver the media into the form in a particulardirection. The direction of the media flow can be designed to betterposition the nerve in the channel. In one embodiment, the hole isprovided adjacent to or on top of where the nerve enters the form toguide the solution from proximal to distal in the conduit and encouragelaminar flow within the form.

Lids. In some embodiments, the form contains a partial or completehinged lid to permit the centering of the nerve according to thedirection of flow within the form.

Volumes delivered. As with the cap form, the volume of media deliveredmay range from 0.1 cc to 10 cc, typically 0.2 to 5 cc, more typically0.3 to 1 cc.

Needle size. The kits contain a 21 gauge or 23 gauge needle fordelivering smaller volumes into smaller size wrap (or cap) forms and 18gauge needles for filling larger forms. These needles provide additionalcontrol to the speed of delivery of the in situ forming material,permitting, the deposition of a bead of the hydrogel to the rapidfilling of a larger conduit.

Gelation time. Similarly, the gelation time can be adjusted depending onthe fill volume of the wrap or cap forms, providing longer gelationtimes of 10 to 20 seconds for larger wraps and a shorter gelation timeof around 5 seconds for smaller wraps or caps.

Form sizes and hydrogel thickness. The range of form sizes are designedwith an entrance region to accommodate a nerve diameter ±1 to 3 mm, ormore preferably ±1 mm. The diameter of the form determines the thicknessof the gel that forms around the nerve. The thickness of the hydrogelthat forms around the nerves may be 0.05 mm to 10 mm, more preferably 1mm to 5 mm, more preferably 1 to 3 mm depending on the size of thenerve.

Kit design. Instead of each kit containing one form for only one size ofnerve, as is the case with implantable nerve conduits and wraps, kitswill contain one to ten, more typically one to cap forms (or wrap forms,or combinations thereof), allowing the physician to select theappropriate size for the procedure as well as the ability to switch theform out without having to request an additional kit. Kits may belabelled according to forms selected—for example forms for a ranges ofnerve sizes, forms for a type of surgical procedure (nerve protector foringuinal repair), or forms appropriate for a specific location of theprocedures (nerve cap for hand surgery, nerve protector for upper limb,nerve form for brachial plexus).

Sheets. In some embodiments, the in situ forming material may bedelivered to a nerve that is placed on a temporary nonadherentbiocompatible sheet such as an Esmarch bandage or other biocompatiblesheet or background (Mercian Surgical Visibility Background Material)routinely used to isolate the nerve from the surrounding tissue. Thegelation time can be shortened to limit the spread of the hydrogelaround the nerve, for example to 10 seconds or less, preferably 5seconds or less. Any excess hydrogel can then be removed from thesurgical site and discarded.

Liquid Cap Form. In another embodiment, the form is not a physical formbut created by the injection of a soluble hydrophobic solution,preferably a viscous solution, such as glycerol. For example, whilesecuring the nerve out of the way, a viscous oil can be delivered to thesurrounding tissue to coat it and prevent the hydrogel from adhering thesurrounding tissue. The solution, if viscous enough, can create arapidly bioerodible form for delivering the in situ forming hydrogel. Inthe preferred embodiment, Solution A is delivered first to block theamine and tissue binding sites and to create a space or region intowhich Solution B can be delivered. In the next step, Solution B isdelivered in the space created by Solution A, or it is delivered in thecenter of Solution B, displacing Solution B away from the site.

No Form. In some embodiments, the space or access does not permit theuse of a form. In some cases, such as in brachial plexus injuries, thesurgical window is so small or the concern of damaging adjacent tissuesis so small that placing a form in the site into which to deliver thehydrogel is not possible. In these instances, the hydrogel may bedelivered directly into a surgical pocket or region in or around thenerve. If the region around the nerve is utilized as a natural form, thecap has an irregular shape that is defined by the boundaries of thetissue on the bottom and sides of the nerve. In one embodiment, sincethe hydrogel is adherent to both the nerve and the tissue, the in situformed material should be carefully peeled off of the muscle and fasciaso that it forms a free-floating bolus in contact with the nerve. Thiswill permit the nerve to continue to move within the region withoutbeing tethered to the surrounding tissue.

In still further embodiments, it is desirable for the hydrogel to takethe shape of the surrounding tissue around the nerve. For example, inembodiments where the nerve is to be ablated and the hydrogel needs tofill the potential space where the nerve is/was and the surrounding areato prevent regeneration. Alternatively, when the hydrogel is deliveredaround visceral nerves where there is frequently a loose and finenetwork of tiny nerve fibers and the space around these nerve fibersneeds to be filled. In another embodiment, the hydrogel fills aroundirregularly shaped nerves or bundles/clusters of nerve fibers and/orcell bodies. In this way, the hydrogel can most effectively delivertherapeutic agents to the region.

Hydrogel placed in a controlled manner in situ around a nerve. Inanother embodiment, however, particularly in dynamic environments inwhich nerves are sliding during motion between muscles, joints, bones,or tendons, such as between muscles in the periphery, it is notdesirable for the nerve to be tethered via the hydrogel to thesurrounding tissue. Instead, it is desirable to develop solutions inwhich the nerve can glide freely within the channel. In theseembodiments, the hydrogel can be physically separated from thesurrounding tissue during in situ crosslinking or in situpolymerization. This can be achieved with something as simple as anonadhesive sterile sheet which can be placed at the site and thenremoved after gel formation. This can also be achieved through theplacement of a form in/around the nerve. The form may take several formsdepending on the size and location of the nerve, the presence or absenceof a sheath, the goal of the therapy that is delivered to the site(nerve stimulation, nerve blocking, nerve ablation, or a barrier tonerve regeneration). In one embodiment, the form is a cap that can begently placed around the end of a nerve and the in situ forming gelinjected into this form in order that it assume the shape of the form.The material in the cap can then be pushed out and the cap form removedand discarded. In yet other embodiments, the cap is biocompatible andthus is not removed. In another embodiment, a half-cylinder (halvedlongitudinally) can be placed underneath the nerve and the in situforming material delivered into the half cylinder. In this same manner,it is possible to deliver a gel circumferentially around the nervewithout the gel developing attachments to the surrounding tissue.

Alternate cap forms. In order to reduce the chances of adhesionsforming, the 3 to 10 mm sections of nerve can be placed inside a syringebarrel (with the luer lock end portion removed) and the plunger pulledback to the appropriate gel distance that is desired on the end of thenerve. The hydrogel is delivered into and around the nerve inside theplunger where the gel sets. After the gel forms, the plunger is gentlydepressed to extrude the nerve encased in the hydrogel. Using thisapproach again, minimal or no sutures are required to avoid additionaldamage or over handling of the nerve. Preferably the syringe barrel hasa lubricious coating.

Laprascopic or endoscopic surgery. The forms may be advanced down achannel during laprascopic or endoscopic surgery and placed under anerve, similar to the approach in open surgery. The form may be foldedto permit transit through smaller conduits and then released at the siteof the procedure. Alternatively, instruments can be designed with thenerve form (cap or wrap) build into the tip of one instrument with thein situ forming biomaterial delivered through the lumen of anotherinstrument. Gelation time of the in situ forming biomaterial needs to beadjusted to 20 to 30 seconds or more to allow for travel time of thehydrogel down the instrument lumen.

In a needleoscopic approach to the procedure, a first material can beinjected that coats the outside tissues to prevent direct adhesionbetween the gel and the surrounding tissue. After this, the hydrogel canbe delivered into the same site, forming a depot around the nerves anddisplacing the first material to the periphery of the injection site.This can be achieved with a hydrophobic substance, such as an oil or aviscous substance such as glycerol. This may also be achieved with a lowmolecular weight PEG solution which has the added benefit of helpingseal the membranes of the nerve prior to the hydrogel forming the nerveblock/cap around the nerve.

In another embodiment, the nerve is dipped in the flowable materialsolution prior to it crosslinking to form a thin protective surface onthe hydrogel. In some embodiments, only a thin coating of thebiodegradable polymer is necessary around the nerve. The coating may beonly 100 microns to 500 microns thick. In other embodiments, a coatingis not sufficient to prevent the inflammatory infiltration and orprevent early degradation—in these cases it is desirable to use acoating of between 0.5 mm to 10 mm thick.

Attempts at developing nerve caps to date have been focused on solidphysical caps that are sutured in place around the end of a transectednerve. These caps have, by necessity, had a gap around the end of thenerve at the end of the proximal stump as well as circumferentially. Asa result, neuroma formation occurs into the end of the cap. Examplesinclude resorbable poly(D,L lactide-co-caprolactone) implant, alignedsilk fibroin (SF) blended with poly(L-lactic acid-co-e-caprolactone)(SF/P(LLA-CL)) nanofiber scaffolds, poly(lactic acid)-co-(glycolicacid)/arginylglyclaspartic acid) modified poly(lactic acid-co-glycolicacid-alt-L-lysine) (PRGD-PDLLA) implant with pores on the order of 10microns in diameter [), Yi et al 2018, Adv Sci]. The PRGD/PDLLA conduitwas 10 mm long with an inner diameter of 2 mm and a tube wall thicknessof 200 microns, the SF/P(LLA-CL) conduit is 1.5 cm long with an internaldiameter of 1.5 mm. These caps require suture placement. Anotherapproach, called Neurocap®, is a synthetic nerve capping deviceincluding of a solid tube with a closed end that is placed over thenerve bundle and then has to be both sutured to the nerve to keep thenerve within the cap and sutured to the surrounding tissue to hold thecap in position, published as WO2016144166A1. Another approach alsoutilizes a solid implant, published as US20140094932A1. In contrast, theinjectable gel approach can flow around nerves of any size from tinyfibers to large nerve bundles, does not require cutting or suturing, andprovide a reduction in pain and neuralgia. In short, an injectableflowable system is not limited to nerve stumps but also can preventaberrant nerve outgrowth in fibers too small to be picked up.

NERVE PROTECTOR/WRAP. In accordance with a further aspect of theinvention, there are provided methods and devices to protect intact orcompressed nerves. In some instances, it may be desirable to protectnerves that are surgically exposed as a result of a procedure oradjacent nerves or tissues, such as in the instance where these nervewould otherwise dry out. In some instances, it may be desirable toprotect and mark the nerves that are exposed as part of another surgeryso that additional handling, stretching, contusion and/or compressioncan be reduced or avoided. In one embodiment, an in situ formingmaterial is delivered around the nerve to provide a protective layer andprevent the nerve from damage from forceps and other surgical equipmentin the region. Additionally, a dye in the hydrogel can providesufficient contrast from the surrounding tissue that the physician canalso visually stay away from the nerve during the procedure. This maydramatically reduce the incidence of iatrogenic nerve injury duringsurgical procedures.

In accordance with a further aspect of the invention, there is provideda form for creating a formed in situ capsule around a nerve to nervejunction. The form comprises a concave wall defining a cavity, the wallhaving a top opening for accessing the cavity. The top opening lies on afirst plane and has an area that is less than the area of a second planeconforming to inside dimensions of the cavity and spaced apart into thecavity and parallel to the first plane. A first concave nerve guide iscarried by the wall and provides a first side access for positioning afirst nerve end in the cavity; and a second concave nerve guide iscarried by the wall and provides a second side access for positioning asecond nerve end in the cavity.

One example of this is the prophylactic treatment of the ilioinguial andiliohypogastric nerves during procedures to repair hernias, particularlyinguinal hernia repair. These nerves may be partially or completelyexposed during the repair of the hernia, resulting in compression,contusion, and partial or complete transection. Post-surgically, thedamaged nerve may send aberrant nerve sprouts out into the post-surgicalscar tissue which may result in neuroma formation and nerve entrapmentresulting in a high rate of post-operative and chronic pain. Inaddition, these nerves may be incidentally or purposefully transectedsurgically in an attempt to prevent the nerves from being entrappedsurgically or tangled in the mesh used to repair the hernia. In oneembodiment, a kit containing the in situ forming growth inhibitoryhydrogel and appropriate forms permit the surgeon to select a form toprovide either a ‘cap’ or ‘wrap’ shaped form cavity. Depending on thesurgery, the physician may then select a wrap if the nerve is nottransected and the physician wishes to protect the nerve from furtherdamage or a cap if the nerve is transected and the physician wishes toprevent the formation of distal neuroma at the end of the transectednerve.

Another example of this is the exposure of the sciatic nerve during hipprocedures. Although the nerve is not the target of these procedures,the nerve is often exposed and placed in traction such that it runs arisk that it be damaged and/or dry out during the procedure. In oneembodiment, a wrap-shaped form is provided to deliver hydrogel aroundthe region of sciatic nerve at risk. For larger nerves, these region maybe 5 to 50 cm or more. Wrap-shaped forms may be provided to span thisentire length or alternately multiple cap forms can be placed in seriesalong the nerve to provide protection. In another embodiment, ananti-inflammatory agent is delivered in the hydrogel in order to reducethe inflammation around the nerve that may result as a result ofpositioning or moving the nerve during the surgery. In anotherembodiment, a local anesthetic is delivered into the hydrogel that isplaced around the nerve.

In another embodiment, the hydrogel is delivered around the nerve toreduce inflammatory neuropathies that may result after surgery,particularly peripheral neuropathy that may result in slowly developingsevere pain and/or weakness in the affected limb. The in situ forminghydrogel may be delivered after open surgery or through a percutaneousimage-guided procedure. For percutaneous ultrasound guided orfluoroscopic delivery, echogenic needles are desirable to confirm notonly depth but location of the needle relative to relevant structures.

In another embodiment, an in situ forming hydrogel is delivered aroundthe nerve in a ‘wrap’ form cavity to form a protective compliant wraparound the nerve. The wrap form cavity is left place, providingadditional support and protection during the surgery, and then the wrapform is removed and discarded after completion of the procedure prior toclosing the site. The hydrogel remains in place is to protect the nerve,prevent aberrant nerve outgrowth, and any scar tissue from infiltratingthe nerve.

Coaptation aid. In some embodiments, a nerve that has undergone directcoaptation with sutures can be placed into a wrap form cavity. Thedirect coaptation site may be filled with an injected growth permissivehydrogel or temporary spacer material (e.g. fibrin glue) that can spreadinto the interstices of the site and the growth inhibitory hydrogeldelivered directly around the anastomoses site using a wrap form cavity.

Wrap or protector form. The wrap form cavity comprises a form with twoentrance zones for the nerve with a variable cavity length around theregion of the nerve that needs protection. In shorter wrap forms, thecavity is designed such that the nerve rests on the entrance zones andthe nerve is ‘floating’ between this region and does not make contactwith the walls of the form. The needle that delivers the in situ forminghydrogel delivers the flowable hydrogel solution into the form,surrounding the nerve, where it forms a protective hydrogel around thenerve. The form prevents the off target spread of the hydrogel toadjacent tissues and maintains a consistent thickness of hydrogel aroundthe nerve.

Longer lengths. In situations where there is a long length of nerve toprotect, a longer wrap form cavity may be utilized with small postsdesigned in the bottom of the form to prop and provide stability to thenerve over longer distances. These posts are then removed when the formis removed, leaving only a small exposure between the nerve and thesurrounding environment at a non-critical location away from theproximal nerve stump tip (if one exists). In yet another embodiment, inwhich it is desirable to protect longer lengths of nerve, the in situforming hydrogel solution may be delivered in multiple layers orregions. In one embodiment, the form is filled in multiple sections inorder to maintain the nerve within the center of the form. In anotherembodiment, a first layer of in situ forming hydrogel is delivered tothe bottom of the form, either with the nerve or without the nervecompletely or partially embedded, and then a second layer of hydrogel isdelivered on top of the first layer in order to completely cover andprotect the nerve.

In one embodiment, kits are provided which contain the appropriatevolume of in situ forming hydrogel to fill the wrap form as well asmultiple mixers and needle components to allow the physician to switchthe mixer-needle tip and continue to deliver more of the media in secondor third applications, as needed.

Protecting anastomoses sites. With the increased use of allograft inaddition to autograft in larger nerve gap repairs, there is an increasedrecognition that aberrant nerve outgrowth from the peripheral nervestump into the surrounding tissue at the nerve-allograft,nerve-autograft, or nerve-conduit junction can result in local pain andreduce the effectiveness of the nerve repair. In addition, thecompliance mismatch between a solid implantable conduit used to securetwo nerve stumps and the nerves themselves may cause friction at theinterface between the nerve and the conduit resulting in additionalaberrant nerve tethering into surrounding tissue. In one embodiment, thedelivery of an in situ forming hydrogel at the interface between theproximal nerve and the allograft or autograft anastomoses, or thedelivery of the hydrogel between the allograft or autograft and distalnerve stump, or similarly at the junction between where the nerve stumpenters and exists the conduit, is anticipated. A smaller volume ofhydrogel delivered either directly or in a shorter wrap form segmentprovides protection of neurite outgrowth and reduction in scar formationand immune cell infiltration into the graft and conduit. Alternatively,a wrap form may span the entire length of the anastomoses to cover theallograft/autograft/conduit in addition to the nerve.

Nerve gliding. Some peripheral nerves undergo a considerable amount ofmotion in the fascial plane in which they reside and thus scarring andtethering of these nerves is particularly painful. For example, themedian nerve in the carpal tunnel or the ulnar nerve location relativeto the elbow, are locations where a provision for gliding is important.With implantable conduits, wraps and protectors, the form of the implantis such that the gliding of the nerves is not enhanced with thebiomaterial and may be further inhibited. In one embodiment, the in situforming hydrogel is delivered as a protector around these nerves topermit the nerves to continue to glide within their fascial plane. Oneway this can be accomplished is by delivering a higher swelling in situforming material that swells significantly, e.g. greater than 30%,preferably greater than 60% outward radially after delivery around anerve such that the nerve can glide within the channel created after thehydrogel reaches equilibrium. In this manner, even though the hydrogeleventually becomes enchased in a thin capsular layer, the nerve itself,within the hydrogel, is free to glide within the channel and is freefrom significant scar tissue, nerve outgrowth into surrounding tissueand also is not compressed in these critical locations.

Indications. The in situ forming hydrogels can also be introducedintraoperatively to aid in the maintenance of a successful microsurgicalanastomosis of donor and recipient nerves using a wrap form. Theyhydrogels can be injected in a wrarp form at the junction of theanastomoses to protect the aberrant inflammatory response, the formationof scar tissue, and aid in the coaption of the donor and recipientnerves. The transfer of a noncritical nerve to reinnervate a moreimportant sensory or motor nerve is known as neurotization. In oneexample, a patient undergoing breast reconstruction after mastectomy canselect autologous flap reconstruction to connect the nerves with thechest wall. Wrap forms can be placed at the junction between theproximal nerve stump and distal nerve tissue to which it is sutured andthe hydrogel delivered to protect the anastomoses sites. This repair maylead to restoration of sensory function and an improvement in physicaland quality of life advancement for women.

Compression repair. In another embodiment, the in situ forming hydrogelscan be delivered in a wrap form around the nerve as a barrier to theattachment of surrounding tissue while the nerve repairs. This approachallows the hydrogel to infiltrate and conform to the nerve and act as areplacement for vein wrapping, in which autologous vein in wrappedaround the nerve in a spiral wrapping technique, providing a barrier toattachment of surround tissue. This also provides an alternative to theAxoGuard Nerve Protector which has to be wrapped around the nervepotentially stretching and damaging it further. The solid nerveprotector requires extensive handling of the nerve with forceps,stretching open the solid wrap to hold it open, and then suturing thenerve to the wrap. Using a soft, conformal, hydrogel based approach, aliquid or viscous liquid may be delivered directly around the nerve in aform with minimal nerve handling. Soft tissue attachments are minimized,swelling is minimized and mechanical support provided by the gel reducesthe tension and stress on the coaption site. Nutrients can diffusethrough the hydrogel network. Also, the hydrogel may reduce thephysician's procedure time.

In one embodiment, the solution in based on hyaluronic acid. In anotherembodiment, the solution is based on a hydrogel slurry (TracelT, BostonScientific).

Distance applied. The hydrogel can be delivered circumferentially aroundthe nerve using a syringe or applicator tip, in this manner, the nervehas protection over the length of the damage. Ideally, the hydrogelwould be applied between e.g. 5 and 15 mm on each site of the damaged ortransected nerve. Volumes administered may be between 100 microlitersand 10 ml, more preferably 0.2 to 3 ml. The syringe contains thehydrogel (or the two precursor components to the hydrogel) can bedesigned with the exact volume to be delivered to allow for controlledautomated delivery of the in situ forming hydrogel. Alternatively, anexcess volume can be provided to allow the individual to use his/herjudgment on how much to deliver around the site. At minimum, thehydrogel should form a cellular barrier approximately 200 microns thickaround the outside of the nerve, although the hydrogel may also bedelivered to fill a site and thus form a circumferential bolus with a 2cm radius around the hydrogel site.

End-to-side Repair. A window in the outer nerve sheath is made and anerve transfer is attached to the side of the nerve. After the suturing,the in situ forming hydrogel is delivered around this to keep these inclose apposition with one another.

Internal neurolysis. After a nerve is stretched or chronicallycompressed, internal scarring and swelling my occur. The outer sheath ofthese nerves may be opened to relieve pressure and assist with bloodflow.

External neurolysis. If nerves have become scarred or develop neuromas,stretching or moving may result in additional nerve damage, pain, andadditional nerve scarring. Neurolysis can be used to remove the scartissue around the nerve without entering into the nerve itself. The insitu forming hydrogel can be delivered around the nerve after theexternal neurolysis to prevent additional nerve scarring and reducepain.

Neurotization. In one embodiment a percutaneous nerve protector isdelivered around the damaged or crushed nerve. In this embodiment, ifapplied within a day to several days after injury, the localinflammatory response can be reduced. In situ forming hydrogels thatare 1) biocompatible, 2) biodegradable or bioerodible, 3) permitdiffusion of nutrients and oxygen into and out of the tissue whilepreventing inflammation, 4) flexible and compliant so that the axons areprotected without being compressed, 5) non or minimally swelling, and 6)prevent fibrous ingrowth to the injury site.

Location. An injectable conformable hydrogel also permits the sameproduct to be delivered to multiple nerve diameters and multiplelocations (between bones, fascia, ligaments, muscle) as the materialwill flow in the region around the nerve.

Delivery location. In some embodiments, the needle location impacts thedelivery of the hydrogel. In some embodiments, the needle delivers thehydrogel directly on top of the target nerve or region. In anotherembodiment, the needle is run distally to proximally to first fill theend of the form and the distal nerve stump and later to fill the rest ofthe conduit.

TMR. In another embodiment, the hydrogels can be delivered around nervethat are reconnected as part of targeted muscle reinnervation (TMR)procedures. Due to frequent the size mismatch between the transecteddonor nerve and the typically smaller denervated recipient nerve, it maybe desirable to apply a hydrogel at the junction to help direct theregenerating fibers into the target receipient motor nerve. For theseindications the hydrogel may be applied directly or within a form.Typically this is performed between a mixed motor and sensory nerve.

Inhibitory drugs to caps and wraps. Depending upon the desired clinicalperformance, the mechanical barrier may be assisted or enhanced by anyof a variety of chemical agents that inhibit or prevent nerve regrowth(sometimes referred to as “anti-regeneration agents”). These agentsinclude inorganic and organic chemical agents, including small moleculeorganic chemical agents, biochemical agents, which may be derived fromthe patient and/or from an external source such as an animal sourceand/or a synthetic biochemical source, and cell-based therapies.Anti-regeneration agents may be applied directly to target tissue priorto or following forming the nerve end. Alternatively, theanti-regeneration agents may be carried within the media where theybecome trapped in the media and are then released over time in thevicinity of the nerve end.

Some specific examples of anti-regeneration agents that may be used inconjunction with some embodiments of the present invention include,among others: (a) capsaicin, resiniferatoxin and other capsaicinoids(see, e.g., J. Szolcsanyi et al., “Resiniferatoxin: an ultrapotentselective modulator of capsaicin-sensitive primary afferent neurons”, JPharmacol Exp Ther. 1990 November; 255(2):923-8); (b) taxols includingpaclitaxel and docetaxel (i.e., at concentrations are sufficientlyelevated to slow or cease nerve regeneration, as lower concentrations ofpaclitaxel may facilitate nerve regeneration; see, e.g., W. B. Derry, etal., “Substoichiometric binding of taxol suppresses microtubuledynamics,” Biochemistry 1995 February 21; 34(7):2203-11), botox, purineanalogs (see, e.g., L A Greene et al., “Purine analogs inhibit nervegrowth factor-promoted neurite outgrowth by sympathetic and sensoryneurons,” The Journal of Neuroscience, 1 May 1990, 10(5): 1479-1485);(c) organic solvents (e.g., acetone, aniline, cyclohexane, ethyleneglycol, ethanol, etc.); (d) vinca alkaloids including vincristine,vindesine and vinorelbine, and other anti-microtubule agents such asnocodazole and colchicine; (e) platinum-based antineoplastic drugs(platins) such as cisplatin, carboplatin, oxaliplatin, satraplatin,picoplatin, nedaplatin and triplatin; (f) ZnSO.sub.4 (i.e.,neurodegenerative factor); (g) latarcins (short linear antimicrobial andcytolytic peptides, which may be derived from the venom of the spiderLachesana tarabaevi); (h) chondroitin sulfate proteoglycans (CSPGs) suchas aggrecan (CSPG1), versican (CSPG2), neurocan (CSPG3),melanoma-associated chondroitin sulfate proteoglycan or NG2 (CSPG4),CSPGS, SMC3 (CSPG6), brevican (CSPG7), CD44 (CSPG8) and phosphacan (see,e.g., Shen Y et al. “PTPsigma is a receptor for chondroitin sulfateproteoglycan, an inhibitor of neural regeneration”, Science, 2009October 23; 326(5952):592-6); (i) myelin-associated glycoprotein (MAG);(j) oligodendrocytes; (k) oligodendrocyte-myelin glycoprotein; and (I)Reticulon-4, also known as Neurite outgrowth inhibitor or Nogo, which isa protein that in humans is encoded by the RTN4 gene (see, e.g., LyndaJ.-S. Yang et al., “Axon regeneration inhibitors, Neurological Research,1 Dec. 2008, Volume 30, Issue 10, pp. 1047-1052) (m) ethanol orglycerol.

Further examples of anti-regeneration agents include agents that inducethe formation of the inhibitory scar tissue, which may be selected fromthe following, among others: (a) laminin, fibronectin, tenascin C, andproteoglycans, which have been shown in inhibit axon regeneration (see,e.g., Stephen J. A. Davies et al., “Regeneration of adult axons in whitematter tracts of the central nervous system,” Nature 390, 680-683 (18Dec. 1997); (b) reactive astrocyte cells, which are the main cellularcomponent of the glial scar, which form dense web of plasma membraneextensions and which modify extracellular matrix by secreting manymolecules including laminin, fibronectin, tenascin C, and proteoglycans;(c) molecular mediators known to induce glial scar formation includingtransforming growth factor.beta. (TGF.beta.) such as TGF.beta.-1 andTGF.beta.-2, interleukins, cytokines such as interferon-.gamma.(IFN.gamma.), fibroblast growth factor 2 (FGF2), and ciliaryneurotrophic factor; (d) glycoproteins and proteoglycans that promotebasal membrane growth (see, e.g., C C Stichel et al., “The CNS lesionscar: new vistas on an old regeneration barrier”, Cell Tissue Res.(October 1998) 294 (1): 1-9); and (e) substances that deactivate Schwanncells. Still other examples of anti-regeneration agents includeSemaphorin-3A protein (SEMA3A) (which may be used to induce the collapseand paralysis of neuronal growth cones) to block regeneration isincorporated into the hydrogels to release approximately 1 μg per dayfor a total of 2 μg over a couple weeks, calcium (which may lead toturning of nerve growth cones induced by localized increases inintracellular calcium ions), f) inhibitory dyes such as methylene blue,and g) radioactive particles. Yet other inhibitory drugs includeciguatoxins, anandamide, HA-1004, phenamil, MnTBAB, AM580, PGD2,topoisomerase I inhibitor (10-HCT), anti-NGF, and anti-BDNF.

Pain and inflammation. The media may additionally include one or moreagents intended to relieve pain in the short-term post-proceduretimeframe where increased pain over baseline may be experienced due tolocal tissue reaction depending upon the ablation procedure. Examples ofsuitable anesthetic agents that can be incorporated into the hydrogelfor this purpose include, for instance, bupivicaine, ropivicane,lidocaine, or the like, which can be released to provide short-termlocal pain relief post-procedure around the treatment regionInflammation and scar tissue in the surrounding tissue can also beminimized with the incorporation of methylprednisolone into thehydrogel.

Growth permissive form. Regarding the examples of FIGS. 5A-5E in someinstances, it is desirable to provide a growth-permissive substancebetween the proximal and distal stump of the nerve to encourage nerveregeneration rather than growth inhibition. In some embodiments, thegrowth permissive substance simply provides a temporary barrier to thegrowth inhibitory gel leaking into the anastomoses site or damaged nervetissue and inhibiting regeneration. In other embodiments, the growthpermissive substance provides a medium through which nerves canregenerate without the need for autograft/allograft or conduit/wrap.

In accordance with a further aspect of the invention, there are providedmethods and devices to encourage guided nerve growth, such as to span agap between two opposing nerve stumps and restore nerve function or tofill a small gap between nerves that have been directly coapted withsutures. The method may comprise the steps of placing a first nerve endand a second nerve end in a first form cavity; Introducing an in situforming growth permissive media into the cavity and into contact withthe first nerve end and the second nerve end to form a junction; themedia changing from a flowable to a nonflowable state. The nerves,coupled together by the in situ formed media, are then removed from thefirst form cavity and placed inside a second larger form cavity; andIntroducing a growth inhibitory media into the second form cavity toencapsulate the junction. The growth inhibitory media changes from aflowable to a nonflowable state, covering the nerves and the growthpermissive media; The second form is then removed and discarded. Inanother embodiment, the first and/or second forms remain in place.

There is also provided a formed-in-place nerve regeneration construct,comprising a growth permissive hydrogel bridge having first and secondends and configured to span a space between two nerve ends and encouragenerve regrowth across the bridge; and a growth inhibiting hydrogeljacket encapsulating the growth permissive hydrogel bridge andconfigured to extend beyond the first and second ends of the growthpermissive region to directly contact the proximal and distal nerves,respectively. In yet another embodiment, growth permissive media isdelivered into an inhibitory form cavity where it undergoes a changefrom a substantially flowable to a nonflowable state. The form remainsin place and provides acts as a growth inhibitory substrate throughwhich nerves cannot regenerate.

Preferably, the growth permissive media is comprised of an in situforming gel, such as a hydrogel and the growth inhibitory media iscomprised of an in situ forming gel, such as a hydrogel. However, thegrowth permissive media may be comprised of an in situ forming gel andthe form into which it is delivered is comprised of an ex vivocrosslinked gel.

In some embodiments it is desirable that the growth permissive hydrogeladheres to the nerve tissue, providing a method to anastamose the tissuewithout the need for sutures. In doing so, the nerve-growth permissivegel-nerve unit can be picked up and handled as one continuous nerveunit, permitting their later placement of the unit in a growthinhibitory hydrogel. In other embodiments, the growth permissivehydrogel can provide a temporary glue lasting approximately half anhour. The glue is strong enough to lightly adhere the two nerves but hascomparable mechanical strength to e.g. fibrin glue.

Tensionless repair. Another advantage of in situ forming hydrogels isthat they can be designed to provide tensionless repair of nerves. Inone embodiment, the wrap form of the conduit is deep enough such thatthe directly repaired/anastamosed nerve ends are placed in the form withthe repaired region detensioned inside the form. When the hydrogel isformed around the detensioned nerve, the nerve-nerve repair is not undertension; any tension is carried by the hydrogel around it. In thismanner, the nerves are not under tension and the hydrogel carries theload in a more evenly distributed way than a suture repair can.

In another embodiment, tensionless repair is additionally provided bythe growth inhibitory hydrogel. In this embodiment, the proximal anddistal nerves are placed in the conduit and the growth inhibitoryhydrogel is delivered at the nerve-conduit interface to prevent nervesfrom escaping out of the conduit and tethering with the surrounding scartissue. In another embodiment, the nerves are purposefully detensionedwithin the growth permissive hydrogel, by creating slack in the nerveswithin the form. In cases where the nerves are directly reanastamosed,care is taken to make sure that the tension, if any, is at the interfacebetween the nerve and the entrance to the form on either side of thewrap form, and that the nerve inside the wrap is slack or free oftension before applying the growth permissive hydrogel prescursorsolution into the wrap. In this manner, the nerve anastomoses,nerve-gel-nerve or nerve-nerve interface is without tension. In thepreferred embodiment, the nerve-growth-permissive hydrogel-nerve unitsits entirely within the cavity of the second nerve form. The deliveryof the inhibitory hydrogel provides additional protection anddetensioning, providing approximately 3 to 10 mm of circumferentialcoverage around the nerve on either side of the injury.

Coverage. In one embodiment, the growth permissive media is locatedsubstantially in between the two nerve ends and does not appreciablycover the outer surface of the nerves. Thus, the diameter of the growthpermissive media closely approximates that of the diameter of the nerve.As a result of the location of the growth permissive media, the growthinhibitory media is delivered around the external or epineurial surfaceof the proximal and distal nerves as well as the growth permissivemedia, covering preferably 10 mm or 5 mm of more of healthy nerve oneither side. This permits the guidance of nerves directly from theproximal nerve stump into the distal nerve stump. The additionalcoverage provides adhesion strength and protection from aberrant nerveoutgrowth at the junction of the proximal nerve-gel.

Color. In one embodiment, the growth permissive hydrogel is one color,such as blue, and the growth inhibitory hydrogel has no color. Inanother embodiment the growth permissive hydrogel is blue and the growthinhibitory hydrogel is green or turquoise.

Preferably, the growth permissive substance is an in situ forminghydrogel. Preferably, the growth permissive substance contains growthinhibitory and growth permissive microdomains. Nerves will naturallypathfind along the growth inhibitory domains and within the growthpermissive domains. Growth permissive hydrogels leveraging the in situforming PEG platform are desirable. These hydrogels may be crosslinkedchemically or using photo-crosslinkable approaches as with thenon-growth permissive hydrogels described above. The in situ growthpermissive hydrogels are preferably more rapidly degrading than thegrowth inhibitory hydrogels, encouraging cellular ingrowth andreplacement of the synthetic matrix with natural extracellular matrix.As a result, preferred PEG hydrogels for these applications are formedthrough the crosslinking of PEG-NHS esters with hydrolytically labileester bonds (PEG-SS, PEG-SG, PEG-SAZ, PEG-SAP), preferably PEG-SS. ThesePEGs can be crosslinked with PEG-amines or trilysines, for example.

Other hydrogels may be selected to provide non-growth permissive zonesof the growth permissive hydrogel including PEG-PPO-PEG, PEG-polyesters(triblocks, deblocks), alginate, agar, and agarose. Other synthetichydrogels include PEG-poly(amido amine) hydrogels, PEO, PVA, PPF,PNIPAAm, PEG-PPO-PEO, PLGA-PEG-PLGA, poly(aldehyde guluronate), orpolyanhydrides. An extensive list of hydrogel matrices that may beadapted for in situ formation is found in Hoffman (2012) Hydrogels forbiomedical applications. Advanced Drug Delivery Reviews, 64: 18-24,incorporated herein for reference. Another soft hydrogel that may besuitable includes the InnoCore Liquid Polymer (LQP) (PCLA-PEG-PCLA)which is a liquid polymer which forms a soft macroscopic depot afterdelivery in vivo and degrades slowly over a period of two to threemoths. Another potentially suitable hydrogel includes a six-armedshar-shaped poly(ethylene oxide-stat-propylene oxide) with acrylate endgroups (star-PEG-A) can be photocured. Other start-shaped PEGs include a6-arm or 8-arm NHS ester PEGs include mPEG-SCM (PEG-NHS: SuccinimidlyCarboxyl Methy elster) and mPEG-SG (PEG-NHS: Succinimidly Glutarateester), PEG-co-poly(lactic acid)/poly(trimethylene carbonate), PEG-NHSand trilysine, PEG-NHS and PEG-thiol, PEG-NHS and PEG-amine, PEG-NHS andalbumin, Dextran aldehyde and PEG-amine functionalized with tris (2aminoethyl) amine. PEG concentration. If PEG is used in the growthpermissive matrix, preferably the PEG concentration in these hydrogelsis preferably between 3 and 8%, more preferably 3 to 5 wt % for theapplications to support nerve extension.

In some embodiments, the growth permissive region is directly conjugatedor chemically linked to the non-growth permissive hydrogel region. Forexample, chitosan may be coupled to the inhibitory region. The chitosanmay be a 100 kDa to 350 kDa molecular weight, more preferably 130 kDa to160 kDa with a 0.85 degree of deacetylation. In another embodiment, aninterpenetrating network of gelatin methacrylamide polymerized with aPEG famework.

Alternative growth permissive matrices. In addition to incorporatingpositively charged matrix components that encourage glial invasion,cellular division, and three-dimensional cellular organization, thegrowth permissive components can also support nerve ingrowth with orwithout the presence of supporting cells. These growth promotingsubstances are applied at a concentration sufficient to support growthbut not at such a high concentration to impact the mechanical propertiesof the hydrogel. Growth permissive hydrogels contain combinations ofnatural growth promoting biomaterials such as natural polymers collagentype I (0.01 to 5 wt %, preferably 0.3-0.5 wt %, 1.28 mg/ml), laminin (4mg/ml), hyaluronic acid, fibrin (9 to 50 mg/ml, strength 2.1 kPa) orsynthetic/semi synthetic polymers such as poly-L-arginine orpoly-L-lysine (0.001-10 wt %). By combining the growth inhibitorydomains above with the growth permissive matrices, growth supportivematrices can be formed. These blends support the 1) creation of a paththrough which regenerating nerves can path find, 2) provide a substrateto which neurites can adhere and Schwann cells can migrate in. In oneembodiment the hydrogel is 8-arm 15K PEG-succinimidyl succinate (PEG-SS)crosslinked with trilysine, containing 5 wt % collagen. In anotherembodiment, the hydrogel comprises an 4% PEG (4-arm 10K PEG-SGcrosslinked with 4-arm 20K PEG-amine) containing 0.01% poly-L-lysine. Byreducing the concentration of the crosslinked PEG solution relative tothe growth inhibitory PEGs used in neuroma blocking applications andincreasing the concentration of the positively charged growth permissivebiomaterial, an in situ forming hydrogel can be created with bothinhibitory and permissive domains to encourage nerve outgrowth.

In another example, a non-growth permissive hydrogel (e.g. crosslinkedPEG hydrogel, alginate, methacryloyl-substituted tropoelastin MeTrohydrogel) may be blended with a growth permissive hydrogel (e.g. fibringelatin-methacryloyl GelM, GelM/PEG or GelMA/MeTro composites) Soucy etal (2018) Photocrosslinable Gelatin-Tropoelastin Hydrogel Adhesives forPeripheral Nerve Repair, Tissue Engineering, PMID: 29580168.Incorporation of polylysine. Polylysine—either the D,L, or L forms, canbe incorporated into the growth permissive hydrogel region. For exampleEpsiliseen (Siveele, Epsilon-poly-L-lysine). The growth permissivehydrogel may be an in situ forming hydrogel comprising chitosan andpoly-lysine (https://pubs.acs.org/doi/10.1021/acs.biomac.5b01150). Thegrowth permissive hydrogel may be an in situ forming hydrogel comprisingPEG and polylsyine (http://pubs.acs.org/doi/abs/10.1021/bm201763n).

PEG+Collagen in Backbone. Alternatively, natural polymers, such as typeI collagen, can be crosslinked with PEG hydrogel (e.g. 8-arm 15K SG)with collagen concentrations ranging from 30 to 60 mg/ml and PEGconcentration at 50 or 100 mg/ml ((Sargeant et al 2012. An in situforming collagen-PEG hydrogel for tissue regeneration. ActaBiomaterialia 8, 124-132 and Chan et al (2012) Robust andsemi-interpenetrating hydrogels from PEG and Collagen for ElastomericTissue Scaffolds. 12(11) 1490-1501.

Other gels. In yet another embodiment, the first growth permissivematerial may comprise a viscous solution, a nanoparticle- ormicroparticle-based gel, a slurry, or a macrogel. In one embodiment afibrin glue can be delivered around nerves. In another embodiment, thesolution is a slurry of biocompatible nanoparticles or microparticlesthrough which nerves can regenerate. In another embodiment, a microgelor modugel is delivered to the site. Microgels are created for stabledispersions with uniform size, large surface area through precipitationpolymerization. Modugels, scaffolds formed from microgels, propertiescan be varied through the degree of crosslinking and scaffold stiffness(Preparation of the gels, including PEG-based hydrogels, can be found inScott et al (2011) Modular Poly (ethylene glycol) scaffolds provide theability to decouple the effects of stiffness and protein concentrationon PC12 cells. Acta Biomater 7(11) 3841-3849, incorporated herein forreference.) In addition, the use of electrically conductive hydrogels,such as piezoelectric polymers like polyvinvylidene fluoride (PVDF)which generates transient surface charges under mechanical strain, maybe beneficial in supporting the growth of nerves through the hydrogel.For example, dendrimers comprised of metabolites such as succinic acid,glycerol, and beta-alanine may be incorporated into the hydrogels toencourage extracellular matrix infiltration (Degoricija et al (2008)Hydrogels for Osteochondral Repair Based on Photocrosslinkable CarbamateDendrimers, Biomacromolecules, 9(10) 2863.

Plain natural hydrogels. In another embodiment, an entirely growthpermissive hydrogel is provided without growth inhibitory microdomains.In one embodiment, a fibrin hydrogel (such as those crosslinked withthrombin) with a lower linear compressive modulus is selected. Numerousother biomaterials have also been demonstrated to support nerveregeneration in 2D and 3D scaffolds and include chitosan,chitosan-coupled alginate hydrogels, viscous fibronectin, collagen typeI (˜1.2 mg/ml), assist in regeneration, fibrin (9 to 50mg/ml),fibronectin, laminin(https://www.ncbi.lnm.nih.gov/pubmed/15978668)), Puramatrix, heparinsulfate proteoglycans, hyaluronic acid (1% sodium hyaluronate viscoussolution), polylysine (poly (D, or L, or D,L) lysine), xyloglucan,polyornithine, agarose (0.5% to 1% w/v) and blends of these materials.Additional growth permissive hydrogels include thermosensitive hydrogelslike chitosan-beta-glycerophosphate hydrogels (C/GP) mixtures. Otherthermosensitive hydrogels include poly (N-isopropylacrylamide)(PNIPAAM). In one embodiment, a poly(propylene fumarate) PPF can beinjected as a liquid and chemically, thermally, or photo cross-linked insitu to form a gel to provide a growth supportive hydrogel. In anotherembodiment, an interpenetrating network of HA and photocrosslinkableglycidyl methacrylate hyaluronic acid (GMHA) provides a growthpermissive substrate. Other growth permissive hydrogels include:crosslinked hyaluronic acid gel (Hyaloglide gel) or ADCON-T/N gel(Gliatech). These materials may be physically or covalently crosslinked.Other scaffold materials may be anticipated for the growth permissiveregion (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5899851/).

Charge. It is well known in the art that nerves prefer to grow on orthrough positive charged surfaces. In some embodiments, the positivecharges are incorporated into the polymer backbone. In otherembodiments, these charges are incorporated in other components, such asextracellular matrix proteins that become trapped in the hydrogel whenit forms in situ.

Incorporation of Adjuvants. In some embodiments, anti-inhibitorymolecules can be incorporated into the hydrogel to improve the growthpermissive environment, such as chondroitinase, which breaks downchondroitin sulfate proteoglycans (CSPGs).https://www.ncbi.nlm.nih.gov/pubmed/20620201 These may be incorporatedwith the polymer powder, diluent, or accelerator depending on thestability requirements of the adjuvant.

Incorporation of Lipid Domains. Lipid domains may be added to thebackbone or side chains or these polymers to encourage nerve outgrowth.Hydrophobic domains may also be incorporated into the backbone of thehydrogel to support nerve ingrowth through soft and hard regions of thehydrogel. In one embodiment, lipids are added to diffuse between polymerchains and act as plasticizers for the polymeric material thatfacilitates chain moving and improves elasticity.

Adhesion strength. The growth permissive media and the growth inhibitingmedia may transform into hydrogels and have sufficient adherence that,once formed, the nerve ends can be picked up and handled with ease. Theadhesive strength of the subsequently formed nonflowable growthpermissive media, though, permits the nerve-gel-nerve unit to be pickedup with forceps. The unit can be gently placed within a second form topermit circumferential delivery of the inhibitory hydrogel. The adhesionstrength also permits good coupling between the nerve end and thehydrogel.

Stiffness. As matrix stiffness and compressive strength of the hydrogelplay a significant role in promoting or inhibiting nerve regeneration,the mechanical properties of the growth inhibitory and growth permissivehydrogels differ substantially. The growth permissive hydrogel issignificantly softer and less stiff to support and encourage theregeneration of nerve growth cones into the media. Gel stiffness (G*,dynes/sq cm) of the growth permissive hydrogel is preferably softer andmore elastic in character with G* less than 800 dynes/sqcm, morepreferably less than 200 dynes/sqcm. In some embodiments, regions ofsoft substrate (100-500 Pa) are placed adjacent to regions of stiffersubstrate (1,000 to 10,000 Pa). The elasticity of this growth permissivesubstrate should preferably be less than 0.1-0.2 MPa, preferably lessthan 1.5 KPa. On the other hand, the growth inhibitory hydrogel providesthe necessary mechanical strength to maintain the coupling andrelationship between the proximal and distal nerve stumps, reduce oreliminate the need for suturing, and potentially permit tensionlessrepair. Nerve extension into the growth permissive matrix is stronglydependent on matrix stiffness, and pore interconnectivity of pores,charge. As the gel degrades, the nerve extension may also be impacted bythe hydrolytic, oxidative, or enzymatic degradation of the matrix. Forthe growth permissive hydrogel, the stiffness of the gel should moreclosely approximate the elastic modulus of the nerve tissue, at or below1 kPa, preferably 200-300 Pa. Swelling. Given the placement of thegrowth inhibitory hydrogel around the growth permissive hydrogel, thegrowth permissive hydrogel swelling must be less than or equal to theswelling of the growth inhibitory hydrogel. Alternatively, the growthpermissive hydrogel must be sufficiently soft that it does not have thestrength to push on the growth inhibitory hydrogel. Porosity. In someembodiments, the growth permissive media comprises a growth inhibitoryhydrogel filled with highly interconnected macroscopic growth permissivepores that provide a channel through which regenerating nerves canpathfind. Pores may created in the hydrogels through porogen leaching(solid, liquid), gas foaming, emulsion-templating to generatemacroporosity. The pores may be created by a growth permissive porogenand/or contain therapeutic agents or simply be filled with saline. Thepores may be created by phase separation during hydrogel formation. Theaverage pore size, pore size distribution, and pore interconnections aredifficult to quantify and therefore are encompassed in a term calledtortuosity. Preferably, the hydrogel is a macroporous hydrogel withpores greater than 1 μm, more preferably greater than 10 μm, preferablybetween >100 μm more preferably >150 μm, with an average pore radius of0.5 to 5%. The density of the pores should be greater than 60%, orpreferably greater than 90% pore volume, of sufficient density that thepores are interconnected. In this manner, the remaining PEG hydrogelprovides a non-growth permissive scaffold through which neurons cangrow. In one embodiment, porosity is created by creating air or nitrogenbubbles in the hydrogel through shaking, pushing the plunger in theapplicator back and forth, or introducing the air through another portin the applicator. In another embodiment, a surfactant is used as anair-trapping agent to create porosity in the hydrogel, such as sodiumdodecyl sulfate (SDS). In situ gas foaming with up to 60% porosity and50 to 500 micron pores and a compressive modulus of 20-40 Mpa, describedin https://www.ncbi.nlm.nig.gov/pmc/articles/PMC3842433/. In anotherembodiment, the creation of a foaming agent which generates macroscalepores to permit cellular migration and proliferation. In someembodiments, the porogen is a degradation enhancer. Preferably, theconcentration of pores is sufficient that the pores are interconnectedwith one another. Preferably, >70% of the pores are interconnected, morepreferably 80% or more. The pores create and define growth permissivezones and preferably the interconnectivity is sufficiently high that thetortuoisity is low that neves will extend out into them. In addition, ifthe walls of the pores are formed of PEG, nerves can pathfind along thewalls of the hydrogel. Pores may be created by low molecular weightend-capped PEGs, such as PEG 3350, which can be delivered in up to 50 wt% solution. The growth permissive regions or pores may contain naturalbiomaterials such as collagen/gelatin, chitosan, hyaluronic acid,laminin (Matrigel), fibrin that provide a growth permissive substratefor nerve outgrowth.

Channels. In another embodiment, channels are created in the hydrogel insitu to permit nerve guidance. In one embodiment, channels areapproximately 150 μm, 300 μm in diameter, more preferably 500 μm indiameter to 1 mm. Preferably, the channels are filled with saline insitu.

Fibers and other structural elements. Adding fibers or structuralelements (e.g. beads, macrospheres, gel particle slurry, microspheres,rods, nanoparticles, liposomes, rods, filaments, sponges) to reinforcethe structural integrity of the hydrogel, improve the hydrogels in vivopersistence and/or to provide a substrate along which neurites canextend and growth for guidance, is desirable. The nanofibers can beflexible or rigid and can range in size from nanometers to micrometersin diameter, and can be linear or irregularly shaped. In the preferredembodiment, the fiber deposition through the needle containing the mediainto the form permits the generally longitudinal parallel alignment ofthe fibers within the conduit. The fiber-loaded media is laid down inthe conduit by first filling the distal end and advancing the needle toproximal end of the form in a smooth continuous motion while depositingthe hydrogel. Rapid gelation (less than 20 seconds, preferably less than10 seconds, more preferably less than 7 seconds) permits the fibers tobe captured in the desired orientation as the media changes to anonflowable form. In another embodiment, the media solutions is moreviscous, between 10 and 20 cP, permitting the suspension of these fiberswithin the growth permissive media. In another embodiment, the fibersare provided in the kit and are placed in the lumen with forceps.

Fibers, rods, filaments, sponges. In another embodiment, the fibers areadded to the form either immediately before or after the delivery of thegrowth permissive hydrogel solution in the wrap form to provide asurface along which nerves can grow en route to the distal stump. Thefibers may be added using forceps, another syringe, or sprinkling themwithin the conduit. The gelation time of the hydrogel media issufficiently delayed that the fibers can be embedded within the mediaprior to the media change to a substantially nonflowable form.

Injecting nanorods Similarly, shorter nanorods may be incorporated intothe polymer solution, polymer powder, diluent, or accelerator and theninjected in situ. By injecting smoothly and in one direction andutilizing a fast gelling hydrogel, the alignment of these fibers may beimproved. The fibers may be constructed from nondegradable orbiodegradable materials. In some embodiments the fibers are made ofchitosan, polycaprolactone, polylactic or glycolic acid, or combinationsthereof. The fibers may be inert of functionalized with a positivecharge or addition of a coating such as laminin.https://www.ncbi.nlm.nig.gov/pubmed/24083073. In another embodiment thefibers undergo molecular self-assembly to form a fiber or cable.

In one embodiment, fibers will be incorporated either randomly or in analigned fashion in order to provide the support for nerve regenerationacross a gel. Filaments and sponges can be formed out of collagen. Rodscan be formed out of collagen-gag, fibrin, hyaluronic acid, polyamide,polyarylonitrile-co-methacrylate, PAN-MA, PGA, PHBV/PLGA blends, PLLA,PLGA or PP. The filaments may be between 0.5 and 500 μm in diameter,more preferably 15 to 250 μm in diameter. In one embodiment, the rods,fibers, and filaments may be coated with laminin.

Nanofibers can be incorporated into the hydrogel to provide structuralsupport. Fibers may be composed of PEG, PGA, PLA, PCL, PCL mixed withgelatin, PCL with a laminin coating, chitosan, hyaluronic acid, gels,hyaluronan, fibrin, or fibrinogen (10 mg/ml). In one embodiment, afibrillar fibrin hydrogel (AFG), or P(D,L, LA) fibers, fabricatedthrough electrospinning is are incorporated into in situ forming gels.(Electrospinning methods are described in McMurtrey (2014) Patterned andfunctionalized nanofiber scaffolds in three-dimensional hydrogelconstructs enhance neurite outgrowth and functional control. J. NeuralEng 11, 1-15, incorporated herein.) In another embodiment, polyethyleneglycol is incorporated as a porogen and nanofibers, such as cellulosenanofibers, are employed to provide structural integrity to the softporous hydrogel (Naseri et al (2016) 3-Dimensional Porous NanocompositeScaffolds Based on Cellulose Nanofibers for Cartilage TissueEngineering: Tailoring of Porosity and Mechanical Performance. RSCAdvances, 6, 5999-6007, incorporated for reference herein.)

Microparticles. In yet another embodiment, microparticles, nanoparticlesor micelles can be introduced into the growth permissive media todeliver drugs to the nerve tissue. In one embodiment, microparticles arecomposed of PEG hydrogels (e.g. 8-arm 15K SG, 10%), poly(D,lysine)microparticles. For example, cross-linked PEG particles formed ex vivocan be formulated into a slurry lubricated by low MW PEG (1-6%, 12 kDa).Alternatively, the particles can be suspended in a collagen orhyaluronic acid solution to provide a growth permissive matrix throughwhich the nerves can regenerate. Similarly, hydrophobic particles andoils may be incorporated to create growth permissive voids in thehydrogel to encourage nerve outgrowth.

Compressive modulus of growth promoting hydrogels. Matching thecompressive modulus of the nervous tissue to the growth permissivehydrogel may also be advantageous—approximately 2.6 to up to 9.2 kPa(Seidlits et al (2010) The effects of hyaluronic acid hydrogels withtunable mechanical properties on neural progenitor cell differentiationare promising (Biomaterials 31, 3930-3940). Similarly, the linearcompressive modulus is less than 20 kPa, preferably less than 10 kPa,more preferably less than 1 kPa to encourage nerve and Schwann cellsingrowth into the gel.

In situ forming growth-permissive hydrogels that can be delivered in awrap form or thin layer around partially transected, compressed, orcompletely transected nerve ends are desirable. The use of an in situforming gel eliminates the need to transect otherwise largely intactnerves and provides a mechanism to support nerve regeneration through asubstrate and into the distal tissue. Coupling the growth-permissivehydrogel with a growth-inhibitory hydrogel assists in guiding anddirecting these neurites within the growth permissive region. In oneembodiment, the in situ forming hydrogel has sufficient adhesivestrength and stiffness that it can be delivered between the nerve stumpsinto an appropriate form and then be picked up and removed from thefirst wrap form and placed into a second larger wrap form into which thegrowth inhibitory in situ forming hydrogel is delivered.

Hydrogel thickness. Growth permissive gel. The thickness or diameter ofthe growth permissive gel should roughly approximate the diameter of thenerves to which it is being delivered. In the case where only a smalldefect exists in the nerves, the growth permissive gel can be droppeddirectly on the injured tissue to form a thin layer. Growth inhibitorygel. Given the often rigorous environment in which a nerve is located,often in a fascial plain between or along muscles, in some embodimentsit is desirable that a minimum thickness of growth inhibitory hydrogelbe maintained around the nerve, preferably 1 mm circumferentially, morepreferably 2-3 mm. For example, for a form that would be place aroundthe common digital nerve, approximately 2-2.5 mm in diameter, a conduitof approximately 3 to 4 mm in diameter is used, providing a 0.5 to 2 mmhydrogel layer around the nerve. For the digital nerve, approximately 1to 1.5 mm, a conduit approximately 2 to 2.5 mm in diameter is selected.For larger nerves embedded in the arm or thigh, between 2 and 10 mm,preferably the gel thickness is 2 to 6 mm, preferably 2 to 3 mm aroundthe nerve circumferentially.

Gelation time. After 30 seconds or less, preferably 20 seconds or less,more preferably 10 seconds or less, more preferably 3 to 7 seconds, thehydrogel forms around the nerve. The hydrogel is transparent so thelocation of the nerve can be visually confirmed in the hydrogel. Theclinician visually or mechanically confirms the hydrogel formation andthe silicone form is slipped off of the hydrogel cap and discarded. SeeFIG. 2. The surrounding tissues (muscles, skin) are then sutured upagain per standard surgical technique.

In Vivo Persistence of Growth Permissive Gel or Slurry. The in vivopersistence may be considerably less in the growth permissive hydrogelthan the growth inhibitory gel, permitting the progressive invasion bySchwann cells and regenerating nerve fibers. For growth permissivehydrogels, more rapidly degrading hydrogel networks are desirable topermit cellular infiltration and subsequent nerve regeneration.Preferably, the hydrogels should degrade in between 2 months and 6months, more preferably 3 months. Degradation of the inhibitory region.The inhibitory guide preferably remains in place for 1 month or more,more preferably 3 months or more, to provide support to the regeneratingnerve. In some embodiments, the degradation of the growth permissivehydrogel is days to months, preferably days to weeks, permitting theclearance of the material as the cellular tissue replaces andregenerates.

Charge. Preferably, the growth permissive hydrogels are positive chargedor contain positively charged domains. Addition of PEG fusogens. In someembodiments, it may be desirable to add a nonreactive fusogen to thehydrogel formulation. Thus, in addition to the mechanical blockingproperties of the hydrogel, the damaged proximal surviving nerves may beprotected from excitotoxic damage and their membranes resealed.Furthermore, the hyperexcitability of the cell bodies is reduced, suchas the dorsal root ganglia, reducing neuropathic paresthesia anddysthesia accompanying nerve injury. In one embodiment, low molecularweight end-capped or nonreactive PEG (methoxy-PEG) to the formulation.For example, the trilysine buffer may contain nonreactive low molecularweight linear PEG (0.2 kDa, 2 kDa, 3.35 kDa, 4 kDa, or 5 kDa). Whenmixed with the 8-arm 15K star-shaped PEG, the resulting hydrogel willhave low molecular weight PEG (2 kDa, 10-50% w/v) which may help to sealup the damaged nerve endings and thus further reduce the influx andefflux of ions. In this manner, lysosome formation, demyelination, andand other membrane debris can be prevented from accumulating at thesite. In another embodiment, cyclosporin A may be applied with thesolution to improve the survival of the ablated axons.

In another embodiment, six-armed star-shaped end-capped PEG(poly(ethylene-oxide-stat-propylene oxide) or star-PEG-OH) can be addedas the fusogen. The linear PEG that is mixed in the polymer blend candiffuse out of the crosslinked network, creating micropores up to onemicron in diameter, facilitating diffusion of nutrients but not neuriteextension. The linear PEG based hydrogels are more stiff than thestar-PEG based fusogen addition. The addition of the linear PEG is basedon findings which had been demonstrated 2 kDa PEG to be beneficial inrapidly restoring axonal integrity, called ‘PEG-fusion’ between nervesof cut- and crushed-axons (Britt et al 2010, J. Neurophysiol, 104:695-703). The theory is that this is in part because of sealing of theplasmalemmal and axolemma at the lesion site.

Reapplying or repositioning. Should the clinician not be happy with thelocation of the nerve, the hydrogel ‘cap’ can be removed with forcepsand the procedure repeated. Nerve sparing. In yet another embodiment, itmay be desirable to deliver the in situ forming hydrogel around a nervein order to reduce the handling of the nerve during a procedure. Bydelivering it in and around the nerve bundle, the hydrogel can set andprevent forceps or any other micromanipulators from crushing it duringthe procedure. In additional to protecting the nerve from mechanicaldamage, the hydrogel may also protect from thermal damage such asthrough cauterizing or RF ablation, cryoablation.

There are several embodiments where existing nerve wraps (e.g. conduitswith a top slit in them that allow the nerve to be pushed into thesemi-rigid wrap) and/or conduits are still desired but the physicianwould like to provide additional support for regeneration either in theform of the application of a growth permissive hydrogel, a growthinhibitory hydrogel, or the combination.

The form for the growth permissive hydrogel is designed to besubstantially the same size as the nerves into which they are placed. Inone embodiment, a silicon form which is a hemi-tube with an innerdiameter approximately equivalent to the outer diameter of the nerves isselected. The nerve are placed in the form either in direct apposition,within close approximation, or, with a gap in order to prevent tension,so that they rest within the form without any tension. The nerves restdirectly on the surface of the form itself for delivery of the growthpermissive hydrogel.

Drugs to Promote Nerve Regeneration. Drugs may be delivered to the nervedirectly prior to the placement of the form. For example, localanesthetic, anti-inflammatory, growth factors agents may be delivereddirectly to the nerve prior to encapsulation with the hydrogel.Alternatively, drugs may be incorporated directly into the hydrogel orincorporated through encapsulation in drug-loaded microspheres,micelles, liposomes, or free-base to achieve an improved sustainedrelease profile.

Drugs for pain relief. In some embodiments, drugs used for the treatmentof chronic neuropathic pain may be delivered in the hydrogels includingtricyclic antidepressants, selective serotonin and noradrenalinereuptake inhibitors, antiepileptics, and opioids. For example,pregabalin and gabapentin may be selected for their analgesicproperties. Similarly, duloxetine, vennlafaxine, the SNRI inhibitor andcombinations thereof to provide more comprehensive pain relief.Anti-inflammatories such as diclofenac may also be promising. Otherpotential targets include ligands for the FK506-binding protein family,neuroimmunophilin ligands, which are neuritotrophic, neuroprotective andneuroregenerative agents.

The local delivery of taxol and cetuximab have also shown promise forimproving the survival and regeneration of neurons and may be suitablefor stimulating nerve regeneration when delivered locally in an in situforming hydrogel. In another embodiment, cyclic AMP (cAMP) or cAMPanalogue dibutyryl cAMP promotes regeneration of nerves and may beincorporated into an in situ forming hydrogel to promote nerveregeneration after injury. In another embodiment, Kindlin-1 andKindlin-2 (fermitin family) and drugs which bind to the integrinsuperfamily of cell surface receptors, allow nerves to extend acrossinhibitory matrix and can be incorporated into the hydrogels to enhanceregeneration across inhibitory extracellular matrix.

In another embodiment, tacrolimus (FK506), an immunosuppressant, may beincorporated into the hydrogel to enhance axon generation and speed. Thefinal concentration of FK506 in the formed hydrogel is 100 ug/ml to 10mg/ml, more preferably 0.1 mg/ml. The drug is released for weeks tomonths, preferably at least a month, more preferably at least 3 monthsto aid in immunosuppression and enhance nerve outgrowth. Drugs includeFK506, drugs selective for selective inhibition of FKBP12 or FKBP51.

Drugs that are P2X receptor antagonists (P2XR), P2X3 receptorantagonists (e.g. AF-219 Gefapivant, AF-130), P2X4 and P2X7 receptorantagonists that are implicated in visceral and neuropathic pain (aswell as migraine and cancer pain), are of interest. P2X7 ReceptorAntagonists. The purinergic receptor antagonist Brilliant Blue G (BBG)and the structurally similar analogue, Brilliant Blue FCF (BB FCF), areof particularly interest for their ability to modulate the nerveenvironment after injury (Wang et al. 2013. The food dye FD&C Blue No. 1is a selective inhibitor of the ATP release channel Panx1. J. Gen.Physiol. 141(5) 649-656)). Other dyes of interest include the FD&C GreenNo. 3 dye which, like BBG and BB FCF, inhibit the ATP release Pannexin1channel with an IC50 between 0.2 and 3 uM. A structurally similaranalogue, Brilliant Blue FCF (BB FCF) otherwise known as FD&C #1(https://pubchem.ncbi.nlm.nih.gov/compound/Acid_Blue_9), has also beendemonstrated to improve nerve survival and regeneration after injurywhen used in combination with a low molecular weight end capped PEG 3350Da (https://www.ncbi.nlm.nig.gov/pubmed/23731685). Similar efficacy hasbeen demonstrated using BBG in rat models of sciatic nerve crush(Ribeiro et al 2017) and ischemia in the myenteric plexus (Palombit etal 2019). In addition, BBG is thought to have anti-inflammatory andanti-nociceptive effects through reducing high extracellular ATPconcentrations and high calcium influxes after nerve damage. In oneembodiment, Brilliant Blue FCF is incorporated into the in situ forminghydrogel. The dye can be incorporated into the polymer vial, thediluent, or the accelerator solutions to yield a final concentration inthe gel of 0.0001 to 5%, preferably 0.001 to 0.25%, more preferably 0.01to 0.02% wt % or approximately 1 to 1000 ppm, preferably 10 to 100 ppm.On a per site anatomic basis, local doses of between 5 μg to as high as25 mg of dye may be delivered in a hydrogel locally. For example, theFD&C #1 dye may be delivered at 0.01% concentration in the hydrogels toreduce neuronal injury after stroke (Arbeloa et al 2012—Referenced inPalmobit et al 2019). By incorporating the dye into the hydrogel, thedye may help improve the survival of the transected axons, reduce thelocal inflammation while the hydrogel provides a barrier toregeneration.

In another embodiment, TRPV1 agonists, such as capsaicin are deliveredto the nerve to deliver a preconditioning injury to the nerve that interm results in a neuroregenerative response downstream to enhance nerveregeneration (PMID:29854941). In one embodiment, capsaicin loadedhydrogels (1 to 8 wt % drug loading) are delivered percutaneously tointact nerves to reduce painful diabetic neuropathy). In anotherembodiment, pifithrin-u or acetyl-L-carnitine is delivered in thehydrogel to reduce and treat chemotherapy-induced peripheral neuropathy(CIPN) by reducing neuronal mitochondrial damage.

In another embodiment, drugs that block the deregulated long non-codingRNAs may also be incorporated into the hydrogels, such as targets ofendogenous Kcna2 antisense RNA. In one embodiment, mitomycin C isincorporated into the in situ forming hydrogel in order to inhibitSchwann cell proliferation and stimulate apoptosis in fibroblasts. Inone embodiment, 0.1 to 5 mg mitomycin C are loaded into the polymerpowder and utilized to form an in situ formed gel with 0.01-0.5 wt %mitomycin C releasing between 0.1 and 0.5 mg/ml are released per day,preferably for 7 days or more. In still another embodiment, Rho Kinase(ROCK) inhibitors or ROK antagonists or Rac1 antagonists may beincorporated, such as ripasudil hydrochloride.

Additional drugs include the anti-inflammatory curcurmin, rapamycin,paclitaxel, cyclosporin A, pyrimidine derivatives (RG2 and RG5) tostimulate remyelination, Axon guidance molecule Slit 3, triptolide,KMUP-1. calcium modulating agents including calcitonin, calciumantagonist nifedipine, nimodipine, nerve growth factor (NGF, 500 ng),insulin-like growth factor (IGF-1), thymoquinone, duloxetine (10-30 mg),melatonin, c-Jun or mTORC1 agonists may help support Schwann celldifferentiation and nerve remyelination, nicotine, andadrenomedullin—used as a neuroprotective and neurotrophic drug.

Example 1. Growth inhibitory hydrogel. Into the vial containing 80 mgPEG with NHS ester reactive group, 80 μg of BB FCF is added to yield a0.1% dye concentration in the PEG hydrogel.

Example 2. Growth inhibitor hydrogel with a fusogen. Into the vialcontaining 80 PEG with NHS ester reactive group, 80 μg of BB FCF and 500mg PEG 3350.

Example 3. Phospholipids are incorporated into the PEG hydrogel, such ascephalin, to improve fusion. Phospholipids are surface activeamphiphilic molecules and can be incorporated as an emulsifier, wettingagent, solubilizer, and membrane fusogen. These may includephosphatidylcholine, phosphatidylethanolamine or phosphatidylglycerol(https://www.ncbi.nlm.nig.gov/pmc/articles/PMC4207189/, incorporated forreference herein).

Example 4. In some embodiments, hydrogels are loaded with amiodaronewith or without the addition of ethanol. For example, 0.1 to 5 wt %loading of amiodarone or more can be achieved. This can also beaccomplished and improved with the incorporation of ethanol into thesolution. For example, 50 to 75% ethanol can be incorporated with 0.25wt % amiodarone to achieve burst release of amiodarone between 3 to 5days. Similarly, 1% amiodarone can be delivered from the hydrogels for aperiod of 30-60 days.

Various other modifications, adaptations, and alternative designs are ofcourse possible in light of the above teachings. Therefore, it should beunderstood at this time that within the scope of the appended claims theinvention may be practiced otherwise than as specifically describedherein. It is contemplated that various combinations or subcombinationsof the specific features and aspects of the embodiments disclosed abovemay be made and still fall within one or more of the inventions.Further, the disclosure herein of any particular feature, aspect,method, property, characteristic, quality, attribute, element, or thelike in connection with an embodiment can be used in all otherembodiments set forth herein. Accordingly, it should be understood thatvarious features and aspects of the disclosed embodiments can becombined with or substituted for one another in order to form varyingmodes of the disclosed inventions. Thus, it is intended that the scopeof the present inventions herein disclosed should not be limited by theparticular disclosed embodiments described above. Moreover, while theinvention is susceptible to various modifications, and alternativeforms, specific examples thereof have been shown in the drawings and areherein described in detail. It should be understood, however, that theinvention is not to be limited to the particular forms or methodsdisclosed, but to the contrary, the invention is to cover allmodifications, equivalents, and alternatives falling within the spiritand scope of the various embodiments described and the appended claims.Any methods disclosed herein need not be performed in the order recited.The methods disclosed herein include certain actions taken by apractitioner; however, they can also include any third-party instructionof those actions, either expressly or by implication. The rangesdisclosed herein also encompass any and all overlap, sub-ranges, andcombinations thereof. Language such as “up to,” “at least,” “greaterthan,” “less than,” “between,” and the like includes the number recited.Numbers preceded by a term such as “approximately”, “about”, and“substantially” as used herein include the recited numbers (e.g., about10%=10%), and also represent an amount close to the stated amount thatstill performs a desired function or achieves a desired result. Forexample, the terms “approximately”, “about”, and “substantially” mayrefer to an amount that is within less than 10% of, within less than 5%of, within less than 1% of, within less than 0.1% of, and within lessthan 0.01% of the stated amount. Furthermore, various theories andpossible mechanisms of actions are discussed herein but are not intendedto be limiting.

1. A method of in situ formation of an implant, comprising the steps of:identifying a nerve; positioning the nerve in a cavity defined by aform; introducing media into the cavity to surround the nerve; andpermitting the media to undergo a transformation from a first,relatively flowable state to a second, relatively non flowable state toform a protective barrier surrounding the nerve; wherein a hydrophiliccharacteristic of the media cooperates with a hydrophobic characteristicof the cavity to facilitate a rapid release of the implant from thecavity following the transformation.
 2. The method of in situ formationas in claim 1, comprising positioning a severed end of a nerve into thecavity, and permitting the media to undergo a transformation state toform a protective barrier surrounding the severed end of the nerve. 3.The method of in situ formation as in claim 1, wherein the implant canbe removed from the cavity by a pull force of no more than about 10 N,applied for no more than about 5 seconds.
 4. The method of in situformation as in claim 1, wherein the implant can be removed from thecavity by a pull force of no more than about 5 N.
 5. The method of insitu formation as in claim 1, wherein the implant can be removed fromthe cavity by a pull force of no more than about 2 N applied for no morethan about 2 seconds.
 6. The method of in situ formation as in claim 1,wherein the implant can be removed from the cavity within about 2seconds, without disrupting attachment between the implant and thenerve.
 7. The method of in situ formation as in claim 1, wherein theintroducing media step includes introducing a first volume of media, andfollowing transformation of the first volume, introducing a secondvolume of media.
 8. A method of in situ formation of an implant,comprising the steps of: identifying an implant formation site;positioning a form having an implant cavity at the site; introducingmedia into the cavity to form an implant precursor; and permitting themedia to undergo a transformation from a first, relatively flowablestate to a second, relatively non flowable state to form the implant;wherein a hydrophilic characteristic of the media cooperates with ahydrophobic characteristic of the cavity to facilitate a rapid releaseof the implant from the cavity following the transformation.
 9. Themethod of in situ formation as in claim 8, wherein the implant is anerve cap for inhibiting neuroma formation around a severed nerve end.10. The method of in situ formation as in claim 8, wherein the implantis a nerve conduit for guiding growth of a nerve.
 11. The method of insitu formation as in claim 8, wherein the implant is a tissue bulkingimplant.
 12. The method of in situ formation as in claim 8, wherein theimplant is a vascular occlusion device.
 13. A method of in situformation of a nerve cap, comprising the steps of: identifying a severedend of a nerve; positioning the severed end into a cavity defined by aform; introducing media into the cavity to surround the severed end; andpermitting the media to undergo a transformation from a first,relatively flowable state to a second, relatively non flowable state toform a protective barrier surrounding the severed nerve end; wherein ahydrophilic characteristic of the media cooperates with a hydrophobiccharacteristic of the cavity to facilitate a rapid release of the nervecap from the cavity following the transformation.
 14. A method of insitu formation of a nerve cap as in claim 13, further comprising thestep of removing the form.
 15. A method of in situ formation of a nervecap as in claim 13, comprising identifying a surgically severed nerve.16. A method of in situ formation of a nerve cap as in claim 13, whereinthe form comprises a nerve guide, and said positioning step comprisespositioning the nerve such that the nerve guide maintains the severedend within the cavity spaced apart from a sidewall of the form.
 17. Amethod of in situ formation of a nerve cap as in claim 16, comprisingpositioning the severed end at least about 1 mm away from the sidewall.18. A method of in situ formation of a nerve cap as in claim 13, whereinthe transformation occurs within about 30 seconds of the introducingstep.
 19. A method of in situ formation of a nerve cap as in claim 13,wherein the transformation occurs within about 10 seconds of theintroducing step.
 20. A method of in situ formation of a nerve cap as inclaim 13, additionally comprising the step of blotting a volume ofexoplasm from the severed nerve prior to the introducing step. 21-112.(canceled)